Antibodies Specific for Hyperphosphorylated Tau and Methods of Use Thereof

ABSTRACT

The present invention relates to a class of monoclonal antibody that specifically binds the phosphorylated serine 396 residue on pathological hyperphosphorylated (PHF) tau (pS396) with improved affinity, as well as to methods of using these molecules and their tau binding fragments in the treatment of Alzheimer&#39;s disease and other tauopathies.

FIELD OF THE INVENTION

The present invention relates to a novel class of monoclonal antibodiesthat specifically binds the phosphorylated serine 396 residue onpathological hyperphosphorylated (PHF) tau (pS396), as well as tomethods of using these molecules and their tau binding fragments in thetreatment of Alzheimer's disease and tauopathies.

REFERENCE TO SEQUENCE LISTING

This application includes one or more Sequence Listings pursuant to 37C.F.R. 1.821 et seq., which are disclosed in computer-readable media(file name: 1049-WO-PCT_FINAL_ST25_1.txt, created on Jul. 6, 2017, andhaving a size of 67,266 kB), which file is herein incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Age-related neurodegenerative diseases such as Alzheimer's disease (AD)and dementia are one of the largest societal challenges today. The WorldHealth Organization estimates that costs for care of the elderly willcontinue to increase and that the number of diagnosed dementia caseswill triple by 2050 (World Health Organization and Alzheimer's DiseaseInternational-Status Report (2012) DEMENTIA: A public health priority,WHO). The first treatments for AD were neurotransmitter modulators suchas acetylcholine esterase inhibitors and NMDA modulators. Thesetherapies became available at the turn of the millennium and still formthe cornerstone for symptomatic relief of memory deficits related todementia and AD. However, these drugs do not target the underlyingcauses of AD: accumulation of amyloid-β (Aβ) peptide and tau proteinaggregates and associated loss of neuronal synapses and eventuallyneurons.

Longitudinal, community-wide studies of the elderly (Weiner, M. W. etal. (2014) ADNI online: http://www.adni-info.org/; Breteler, M. M. etal. (1992) Neuroepidemiology 11 Suppl 1, 23-28; Launer, L. J. (1992)Neuroepidemiology 11 Suppl 1, 2-13) together with large genome-wideassociation studies (Lambert, J. C. et al. (2013) Nat. Genet. 45,1452-1458) have shown that AD is a heterogeneous mix of dementias whereup to 10 percent of the advanced AD patients lack amyloid pathology(Crary, J. F. et al. (2014) Acta Neuropathol. 128, 755-766).Furthermore, seminal pathological studies by Braak & Braak (Braak, H.and Braak, E. (1996) Acta Neurol. Scand. Suppl 165, 3-12) demonstrated aclear correlation between the degree of neurofibrillary tangle pathologyand cognitive state prior to autopsy. These observations have beenreinforced by several investigators (Nelson, P. T. et al. (2012) J.Neuropathol. Exp. Neurol. 71, 362-381), and in recent longitudinalbiomarker studies, which indicate that cerebrospinal fluid (CSF) levelsof tau and hyperphosphorylated tau increase throughout early and latestages of the disease (Jack, C. R., Jr. et al. (2013) Lancet Neurol. 12,207-216).

As indicated above, the microtubule-associated protein, tau, and itshyper-phosphorylated version, form the main constituent of intracellularneurofibrillary tangles, which are one of the main hallmarks of AD.Furthermore, specific genetic variants of tau are associated withfamilial forms of fronto-temporal dementia (FTD). Appearance of taupathology in AD occurs in a distinct spatial pattern, starting in theentorhinal cortex, followed by hippocampal and cortical areas (Braak, H.and Braak, E. (1996) Acta Neurol. Scand. Suppl 165, 3-12). The specificstage of tau pathology also correlates well with cognitive abilities(Nelson, P. T. et al. (2012) J. Neuropathol. Exp. Neurol. 71, 362-381;Braak, E. et al. (1999) Eur. Arch. Psychiatry Clin. Neurosci. 249 Suppl3, 14-22). Taken together, this evidence forms the basis of a tau-basedhypothesis for AD. It entails that the intracellular accumulation of tauleads to microtubule degeneration and spinal collapse. As a result,communication between neurons malfunctions and cell death follows.Recently, it has also been shown that tau itself may form anendo-pathogenic species that can transmit neurodegeneration from onecell to the next (Clavaguera, F. et al. (2009) Nat. Cell Biol. 11,909-913).

I. Tau as an Endo-Pathogen

Clavaguera and colleagues have demonstrated that tau itself may act asan endo-pathogen (Clavaguera, F. et al. (2009) Nat. Cell Biol. 11,909-913). Low spin brain extracts were isolated from P301S tautransgenic mice (Allen, B. et al. (2002) J. Neurosci. 22, 9340-9351),diluted and injected into the hippocampus and cortical areas of youngALZ17 mice. The ALZ17 mouse is a tau transgenic mouse line which onlydevelops late pathology (Probst, A. et al. (2000) Acta Neuropathol. 99,469-481). The injected ALZ17 mice quickly developed solid filamentouspathology, and administration of tau immuno-depleted brain extracts fromP301S mice or extracts from wild type mice did not induce tau pathology.Fractionation of the brain extracts in soluble (S1) andsarcosyl-insoluble tau (P3) (Sahara, N. et al. (2013) J. Alzheimer's.Dis. 33, 249-263) and injection of these into ALZ17 mice demonstratedthat the P3 fraction is most competent in inducing pathology. Itcontains most of the intracellular hyper-phosphorylated filamentous tau.The majority of pathology could also be induced when injecting P301Sextracts into the brains of wild type mice, but no NFTs were formed. Insubsequent studies, Clavaguera et al. have shown that human tauextracted from post-mortem brain tissue of other tauopathies(Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP),and Corticobasal Degeneration (CBD)) may also induce tau pathology inthe ALZ17 model (Clavaguera, F. et al. (2013) Proc. Natl. Acad. Sci.U.S.A. 110, 9535-9540). Since the presentation of these data, severalother tau seeding and spreading models have been reported (Ahmed, Z. etal. (2014) Acta Neuropathol. 127, 667-683; Walker, L. C. et al. (2013)JAMA Neurol. 70, 304-310). The main conclusion from these studiesindicates a mechanism by which pathogenic tau in intracellularinclusions is secreted from the cell into the periplasmic space. Thepathological tau material is then transported along the vesicular sheathin both anterograde and retrograde direction and subsequently taken upby neighboring cells by means of bulk endocytosis. This mechanismexplains why the spread of pathology observed in human disease follows adistinct anatomical pattern. Intriguingly, peripheral administration ofpathological tau may accelerate the formation of tau pathology in ALZ17mice (Clavaguera, F. et al. (2014) Acta Neuropathol. 127, 299-301). Thisspreading mechanism may explain disease propagation in otherproteinopathies (Goedert, M. et al. (2010) Trends Neurosci. 33, 317-325;Sigurdsson, E. M. et al. (2002) Trends Mol. Med. 8, 411-413).

II. Tau Species

The discovery that the tau protein may act as an endo-pathogen hasspawned a search for “The Pathogenic Species” that could be targeted inpotential interventive therapies.

The microtubule-associated protein tau gene (MAPT) is located onchromosome 17 of the human genome and expresses six isoforms of the tauprotein in adult human brain. These isoforms arise from the alternativesplicing of exons 2, 3 and 10 of the 16 exons within the MAPT gene.Exons 2 and 3 express a 29-amino acid repeat and exon 10 expresses anadditional microtubule binding domain. As a result, tau isoforms willcontain 0, 1 or 2 N-terminal repeats and 3 or 4 C-terminal microtubulebinding domains (3R or 4R tau). Commonly six isoforms of tau areexpressed. The longest (2N4R) and shortest (0N3R) isoforms consist of441 and 352 amino acids, respectively (Kolarova, M. et al. (2012) Int.J. Alzheimers. Dis. 2012, 731526). The N-terminal projection domain oftau (2N4R) consists of a 44-amino acid glycine-rich tail and residues45-102 encompass two highly acidic regions (N1, N2-domains). Twoproline-rich regions are found at residues 151-243 (P1, P2 domains). Theremainder of the protein is constituted by four microtubule bindingdomains (R1-R4), followed by a short C-terminal region.

Tau is soluble and highly phosphorylation-labile protein. Approximately20 percent or 85 amino acid residues in the longest isoform of tau arepotential (Ser, Thr or Tyr) phosphorylation sites. Approximately half ofthese have been observed to be phosphorylated experimentally (Hanger, D.P. et al. (2009) Trends Mol. Med. 15, 112-119; Hasegawa, M. et al.(1992) J. Biol. Chem. 267, 17047-17054), and the phosphorylation sitesare clustered around the terminal residues of the microtubule bindingdomains. Tau is dynamically phosphorylated and de-phosphorylated duringthe cell cycle. It must dissociate from microtubules to allow formitosis to occur. Its main role in post mitotic cells (thedifferentiated neuron) is to act as a microtubule stabilizer, allowingfor optimal axonal transport. It can only associate with microtubules inits mostly de-phosphorylated form, thus phosphorylation acts as a directmicrotubule association/dissociation switch within the neuron. Undernormal conditions, cytosolic tau contains on average two phosphorylatedsites. In paired helical filamentous material, at least 7-8 sites arephosphorylated (Hanger, D. P. et al. (2009) Trends Mol. Med. 15,112-119; Hasegawa, M. et al. (1992) J. Biol. Chem. 267, 17047-17054).Hyperphosphorylated, paired helical filamentous tau is a key hallmark ofAlzheimer's disease (Kosik et. al. (1986) PNAS, 86, 4044-4048), adistinct mobility shift of hyperphosphorylated tau is observed inimmune-cytochemical analysis of human AD brain material.

It has been difficult to study the tau protein with traditionalstructural techniques like x-ray crystallography or NMR spectroscopy,reflecting its meta-stable nature. Such studies have mainly beenconducted on domain fragments of the un-phosphorylated tau protein. Theonly structural study to date on full-length tau (2N4R), using NMRspectroscopy, reveals that the protein contains only sparse stretches ofstable secondary structure (Mukrasch, M. D. et al. (2009) PLoS. Biol. 7,e34). This analysis indicates that the secondary structure of thepeptide backbone has a large propensity for adapting a β-sheetstructure. The backbone's first 200 residues are considerably moreordered than the C-terminus encompassing the microtubule bindingdomains. The presence of many specific long-range interactions withinthe protein in solution indicates that it exists in a largely disorderedmolten globular state (Ohgushi, M. and Wada, A. (1983) FEBS Lett. 164,21-24).

Protease products of tau generated in particular by caspase and calpain(Asp13, Glu391 and Asp421) have been identified in tangle material(Gamblin, T. C. et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100,10032-10037). In particular, the truncation at Asp421 has been studiedin detail using the tau C3 antibody, which binds to the free Asp421terminus. This truncation has been postulated as an early event in ADpathogenesis associated with induction of apoptosis (deCalignon A. etal. (2010) Nature 464, 1201-1204). The N-terminal cleavage at Asp13 andthe C-terminal cleavage at Glu391 are considered late events in thepathogenesis (deCalignon A. et al. (2010) Nature 464, 1201-1204;Delobel, P. et al. (2008) Am. J. Pathol. 172, 123-131). Recently, anadditional N-terminal fragment (residues 1-224) was identified in CSFfrom AD and PSP patients, and has been hypothesized to be an earlymarker of disease and particularly pathogenic (U.S. Ser. No. 14/092,539;Bright, J. et al. (2014) Neurobiol. Ageing, 1-17). A similar calpaincleaved fragment was reported by other groups (Ferreira, A. and Bigio,E. H. (2011) Mol. Med. 17, 676-685; Reinecke, J. B. et al. (2011) PLoS.One. 6, e23865).

Apart from hyper-phosphorylation and tau fragmentation,post-translational acetylation (Cohen, T. J. et al. (2011) Nat. Commun.2, 252; Min, S. W. et al. (2010) Neuron 67, 953-966) and O-GIcNAcylation(Zhu, Y. et al. (2014) J. Biol. Chem.) have been proposed to bepathology defining processes in the formation of tangle pathologyassociated with AD.

III. Tau Immunotherapies

Immunotherapies are traditionally separated into passive and activevaccine approaches. In an active vaccine approach, a pathogenic agent oran inactivated pathogenic form thereof, is injected into the patient andthe immune system elicits an immune response. This triggers thematuration of B-cells generating high affinity antibodies or cellularresponse against the administered antigen. In a passive vaccineapproach, the triggering of the immune system is circumvented byinfusing a specific antibody against the antigen. The inherent clearancesystem then removes antibody-bound ligand.

AC Immune is pursuing a mouse monoclonal antibody against phospho-serine409 of tau. Antibodies were profiled against human AD and control braintissue and were selected based on their ability to recognize tanglepathology. The humanized version of two antibodies, hACl-36-2B6-Ab1 andhACl-36-3A8-Ab1, both bind to a tau epitope within amino acids 401-418(WO 2013/151762).

The group of Roger Nitsch has isolated tau auto-antibodies from elderlyhealthy individuals with no sign of degenerative tauopathy. A number ofantibodies have been isolated using full length recombinant human tau(2N4R) to find tau specific antibodies. These were then screened fortheir ability to discriminate tau isolates from diseases and healthyindividuals. Three lead antibodies, 4E4, 4A3 and 24B2, have beendescribed in the patent literature (WO2012049570; US2012087861). Theirepitope mapping indicates that all recognize amino acids within andC-terminal to the microtubule binding region, from position V339 toK369. These antibodies do not exhibit any phospho-specificity.

C2N Diagnostics focuses mainly on developing diagnostic tools for earlydetection of neurodegenerative disease. Antibodies were generatedagainst full length human and mouse tau protein. Eight and fiveantibodies were identified, recognizing human and mouse tau,respectively (Yanamandra, K. et al. (2013) Neuron 80, 402-414). Threeantibodies with different binding kinetics were selected for in vivoevaluation. Namely, HJ9.3, HJ9.4 and HJ8.5, recognizing tau residues306-320, 7-13 and 25-30, respectively, with the last one (HJ8.5) beingspecific for human tau. The antibodies were also selected based on theirability to prevent transfer of pathology in an ingenious mechanisticreporter assay of trans-cellular propagation of tau (Sanders, D. W. etal. (2014) Neuron 82, 1271-1288; Kfoury, N. et al. (2012) J. Biol. Chem.287, 19440-19451). Their evaluation in chronic i.c.v. injection studiesin P301S transgenic mice demonstrated their ability to reduce levels ofhyper-phosphorylated tau protein as determined in immuno-histochemicalanalysis of the treated mice.

The antibodies of Peter Davies were developed originally as diagnostictools that could differentiate between pathological and normal tau in ADand control brain material (Greenberg, S. G. and Davies, P. (1990) Proc.Natl. Acad. Sci. U.S.A. 87, 5827-5831). Evaluation of the therapeuticutility of the PHF1 and MC1 antibodies was demonstrated in P301S andJPNL3 (P301L) (Boutajangout, A. et al. (2011) J. Neurochem. 118,658-667; Chai, X. et al. (2011) J. Biol. Chem. 286, 34457-34467;D'Abramo, C. et al. (2013) PLoS. One. 8, e62402 mice). PHF1 recognizes alinear phospho-tau epitope (pS396, pS404) whereas MC1 is aconformation-dependent antibody that recognizes a structural tau epitoperequiring two distinct parts of the linear sequence, an epitope withinresidues 46-202 and a C-terminal epitope between residues 312-342(Jicha, G. A. et al. (1997) J. Neurosci. Res. 48, 128-132). Injection ofthese two antibodies in chronic 12-13-week immunization studies resultedin substantial reduction of spinal cord and brainstem pathology amongother brain regions, which translated to an attenuation of the motordeficit observed in these mice. (D'Abramo, C. et al. (2013) PLoS. One.8, e62402).

iPerian/Bristol Meyers Squibb has developed tau antibodies against apostulated pathological tau species, composed of an N-terminal fragmentof tau (etau: residues 1-224), which promoted hyperactivity in inducedpluripotent stem cell based neuronal cultures. A portfolio of antibodieshas been developed, but characterization has focused on antibodiesIPN001 and IPN002 that recognize an N-terminal epitope within residues9-18. Accordingly, these antibodies detect elevated tau levels in CSFfrom staged AD and PSP patients that may be an early sign of disease. Invivo injections of the antibodies in JPNL3 (P301L) mice led to partialreversal of progressive motor deficits (U.S. Ser. No. 14/092,539).

Einar Sigurdsson reported the first program to demonstrate the efficacyof tau-based immunotherapy. An active vaccine consisting of tau peptide379-408[pS396, pS404] together with Adju-Phos adjuvant was used toimmunize JPNL3 (P301L) mice. In this study a prominent reduction of taupathology was observed in the vaccine treated mice when compared tocontrol animals. An attenuation of tauopathy-related motor phenotype wasdetected as well. Its efficacy was confirmed in a different mouse model(htau/PS1) not driven by mutant tau (Boutajangout, A. et al. (2011) AAIC2011 (7, issue 4, Supplement edn) p. s480-s431; Congdon, E. E. et al.(2013) J. Biol. Chem. 288, 35452-35465; Gu, J. et al. (2013) J. Biol.Chem. 288, 33081-33095).

Prothena has evaluated three tau antibodies in the K3691 (K3) transgenictau mouse and in a P301L mouse model. Antibodies with varying propertieswere selected for in-vivo evaluation. Two pS404-specific antibodies withdifferent isotype (IgG1/k and IgG2a/k) or a total (pan) anti-tauantibody (IgG1/k) were injected in a chronic paradigm. K3691 mice weretreated with weekly injections for 21 weeks starting at 3 weeks of age,and P301L mice were treated for 7 months with weekly injections startingat 4 months of age. A reduction in tau-positive neurofibrillaryinclusions was observed in the K3 mice with the IgG2a/k pS404 antibody.Both of the pS404-specific antibodies were able to reduce the level ofpS422-positive tau, whereas no reduction was observed in the pan tauantibody treated mice. These studies suggest that: 1) tau clearance maybe antibody isotype-dependent, and; 2) It may be important to target atau species that is relevant to disease, as the total-anti-tau antibodywas unable to reduce hyper-phosphorylated tau (PCT/US2014/025044).

The inventors of the present invention have surprisingly foundantibodies specific for the phosphorylated tau serine residue 396(pS396) to be effective in disease models; this is in contrast to theprior art antibodies which recognize primarily the tau proteinsphosphorylated at both 396 and 404 residues, phosphorylated at the 404residue only or at other residues on tau.

The inventors have developed antibodies which furthermore have aremarkable specificity and selectivity to human pathological tau. Theantibodies of the present invention show a much higher degree ofspecificity and selectivity towards human pathological tau overnon-pathological tau compared to the antibodies of WO2013/050567 (seeFIG. 1 of WO2013/050567). The antibodies of WO2012/045882 reported tohave a specific binding, were elicited from 6 to 9 residue amino acidsequences of Tau amino acids 393-401, 396-401, 394-400 and 393-400. Thiscontrasts from the antibodies of the present invention which wereelicited against pathogenic hyperphosphorylated tau comprising a longeramino acid sequence as described herein.

Furthermore, the antibodies and epitope-binding fragments thereof, ofthe present invention show many advantageous features such as theability to discriminate between pathological and non-pathological humantau protein, and in particular to bind tau associated with Alzheimer's(AD) pathology. In electrophysiological studies, the antibodies, andepitope-binding fragments thereof, of the invention were additionallyable to reverse reduced paired pulse facilitation and spontaneousminiature excitatory synaptic current (mEPSC).

SUMMARY OF THE INVENTION

The present invention relates to monoclonal antibodies, andepitope-binding fragments thereof, capable of specifically binding tothe phosphorylated residue serine 396 of human (2N4R isoform) tau (SEQID NO:1). The antibodies are further characterized by their ability todiscriminate between phosphorylated residues 396 and 404 such that theysubstantially do not bind the phosphorylated 404 residue.

The antibodies of the present invention are selective for pathologicaltau in the presence of non-pathological—yet phosphorylated—tau. Theantibodies of the present invention are able to selectively deplete tautangles of pathological tau in the presence of normal tau. Without beingbound to a particular theory, it is believed that depleting tangles oftau comprising tau protein that has been phosphorylated at tau position396 prevents seeding of pathological tau into tau tangles. Accordingly,one aspect of the invention relates to an antibody that is capable ofselectively binding to 396-phosphorylated tau even when such moleculesare in the presence of tau protein that has been phosphorylated at tauposition 404. A related aspect of the invention relates to an antibodythat is capable of selectively binding to 396-phosphorylated tau evenwhen such molecules are in the presence of non-pathogenic tau. Furtherdefined, the invention relates to an antibody selective for pathologicaltau said pathological tau being hyperphosphorylated tau appearing as 64kDa band (by Western Blot analysis) in transgenic mice overexpressingthe human 2N4R isoform of tau.

One aspect of the invention is directed to an anti-tau antibody that,when used with immune-depleted rTg4510 extracts from transgenic mice,specifically reduces the hyperphosphorylated tau 64 kDa and 70 kDa bandsby at least 90%, while reducing the 55 kDa tau band by no more than 10%.A further aspect of the invention is directed to an anti-tau antibodythat specifically reduces the hyperphosphorylated tau 64 and 70 kDabands by at least 90%, while reducing the 55 kDa tau band by no morethan 10%; or the capability, when used as described herein with extractsfrom human AD post-mortem brains, to specifically reduce the pS396hyperphosphorylated tau bands by at least 90%, while not reducing thenon-hyperphosphorylated tau bands by more than 10%.

Another aspect of the invention is directed to a method of treating apatient with a taupathy, such as Alzheimer's Disease, comprisingdepleting a tangle or attenuating the progression of said tangle, saidtangle comprising hyperphosphorylated Tau, said method comprisingcontacting hyperphosphorylated Tau with an antibody of the inventionsuch that the tangle is depleted, reduced in its content ofhyperphosphorylated tau or progression of tangle formation isattenuated.

Alternatively defined, the invention relates to a method of treating apatient with a taupathy, such as Alzheimer's Disease, said methodcomprising contacting tangles with an antibody selective for tau havingresidue 396 phosphorylated such that the tangle is depleted ofhyperphosphorylated Tau.

One aspect of the invention is directed to a monoclonal antibody tohyperphosphorylated human tau, or epitope-binding fragment thereof,comprising:

-   -   (a) a Light Chain CDR1 comprising the amino acid sequence        selected from the group consisting of SEQ ID NO:3, SEQ ID NO:31;        SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:35; SEQ ID        NO:36; SEQ ID NO:37; SEQ ID NO:38; SEQ ID NO:40; and SEQ ID        NO:46;    -   (b) a Light Chain CDR2 comprising the amino acid sequence of SEQ        ID NO:4; SEQ ID NO:41; and SEQ ID NO:47;    -   (c) a Light Chain CDR3 comprising the amino acid sequence of SEQ        ID NO:5; SEQ ID NO:42; and SEQ ID NO:48;    -   (d) a Heavy Chain CDR1 comprising the amino acid sequence of SEQ        ID NO:6; SEQ ID NO:43; SEQ ID NO:49; SEQ ID NO:52; and SEQ ID        NO:55;    -   (e) a Heavy Chain CDR2 comprising the amino acid sequence        selected from the group consisting of SEQ ID NO:7; SEQ ID NO:28;        SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:44; SEQ ID NO:50; SEQ ID        NO:53; and SEQ ID NO:56; and    -   (f) a Heavy Chain CDR3 comprising the amino acid sequence        selected from the group consisting of SEQ ID NO:8, SEQ ID NO:39;        SEQ ID NO:45; SEQ ID NO:51; SEQ ID NO:54; and SEQ ID NO:57.

One aspect of the invention is directed to a monoclonal antibody tohyperphosphorylated human tau, or an epitope-binding fragment thereof,comprising:

-   -   (a) a Light Chain selected from the group consisting of SEQ ID        NO:12; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:19;        SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22 and SEQ ID NO:23; and    -   (b) a Heavy Chain selected from the group consisting of SEQ ID        NO:11; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:24;        SEQ ID NO:25; SEQ ID NO:26; and SEQ ID NO:27.

A further aspect of the invention is directed to a monoclonal antibodyto hyperphosphorylated human tau, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

An interesting aspect of the invention is directed to a monoclonalantibody to hyperphosphorylated human tau, or epitope binding fragmentthereof, comprising:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:31;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

An interesting aspect of the invention is directed to a monoclonalantibody to hyperphosphorylated human tau, or epitope binding fragmentthereof, comprising:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:31;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39;and further comprising at least one of(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;and(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7.

The antibodies, and epitope-binding fragments thereof, of the inventioncan be used in treating tauopathies such as Alzheimer's disease (AD),Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP),Corticobasal Degeneration (CBD), TBI (traumatic brain injury, mild,acute or chronic), and chronic traumatic encephalopathy (CTE).

The antibodies and epitope-binding fragments thereof of the inventionare furthermore intended for use in treating Psychosis, particularlyPsychosis due to AD or Psychosis in patients with AD, and apathy due toAD or apathy in patients with AD.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Fluid Phase inhibition assay for AD-P3 capture using humanisedC10-2 and variants (C10-2_N32S and C10-2_N32S_A101T). As described inExample 3A, concentration dependent inhibition of AD-P3 capture byP396-specific antibodies hC10-2 (squares), hC10-2_N32S (black circles)and hC10-2_N332_A101T (open circles) was investigated. AD-P3 fractionwere incubated 60 min. at room temperature (r/t) with increasingconcentrations of antibodies (0-1000 nM) prior to incubation with 200ng/ml mouse 010-2 antibody immobilized on 96 well plates. Captured AD-P3antigens were detected with sulfo tagged anti-total tau antibody (MSD).

The 1050 of the hC10-2_N32S (black circles) and hC10-2_N332_A101T (opencircles) antibodies are calculated to be 44 nM and 14 nM, respectively.This is a notable improvement over hC10-2, as can be seen when comparingthe curves of FIG. 1. Accordingly, in one aspect of the invention, theantibodies inhibit AD-P3 in the fluid phase inhibition assay describedherein, such that the signal is reduced by 50% at a concentration of 100nM or less of the antibody based on fluid phase inhibition assay forAD-P3 capture.

FIG. 2. Peptide inhibition assay illustrating apparent affinity hC10-2and related variants. As described in Example 3B, concentrationdependent inhibition of antibody binding in fluid phase solution to Ptau386-408 (pS396) peptide was investigated with antibodies hC10-2(squares), hC10-2_N32S (black circles) and hC10-2_N32_A101T (opencircles). The antibodies were pre-incubated at 1 ng/ml for 60 min. atr/t with increasing concentrations (0-10000 nM) of Ptau 386-408 (pS396)prior to incubation in wells coated with 100 ng/ml Ptau 386-408(pS396/pS404). Well-bound antibody was detected with sulfo-taggedanti-human IgG antibody (MSD).

As can be seen from FIG. 2, Antibody hC-10.2 (1050=24 nM), AntibodyhC10.2_N32S (1050=50 nM) and Antibody hC10.2_N32S, A101T (1050=34 nM)have IC50s of less than 100 nM, and even less than 60 nM, based onapparent affinity studies using fluid phased solution with Ptau (P396)386-408.

FIG. 3 (Panels A-Z and AA-AG). Immunohistochemical detection ofpathological tau in post mortem brains from donors with AD and inrTg4510 mouse brain. As described in Example 4, in the prefrontal cortexfrom 3 different AD donors, hC10-2, hC10-2_N32S and hC10-2_N32S_A101Tlabelled neurofibrillary tangles, neuropil threads and dystrophicneurites. The strongest staining intensities were detected with thehighest concentrations of antibody. Control brain sections are devoid ofimmunoreactivity. All 3 antibodies labelled phosphorylated tau inrTg4510 brain with advanced pathology.

Staining increased from hC10-2 to hC10-2_N32S and to hC10-2_N32S_A101T.The strongest staining intensities were detected with hC10-2_N32S_A101T,then hC10-2_N32S, then hC10-2. At concentrations as low as 100 ng/mLhC10-2_N32S_A101T and hC10-2_N32S, there was immunohistochemicaldetection of pathological tau in Alzheimer's brains.

FIG. 4 (Panels A-F). Decoration of tau structures in rTg4510 micetreated with hC10-2. hC10.2 was administrated i.v. (Panels A, C, E and Frepresent rTg4510; Panels B and D represent tTA). The mice received asingle injection of a volume of 150 μL of hC10-2 antibodies at aconcentration of 80 mg/kg. Brain slices were taken after 3 daysaccording to the process described in Example 5. hC10-2 specificallylabels target structures in vivo in hippocampus and cortex in rTg4510brains, but not in control tTA brains. Paired images for AlexaFluor488and Hoechst signal are shown in hippocampal sections.

FIG. 5 (Panels A-F). Decoration of tau structures in rTg4510 micetreated with hC10-2_N32S. (Panles A, C and E represent rTg4510; PanelsB, D and F represent tTA). The mice received a single injection of avolume of 150 μL of hC10-2_N32S antibodies at a concentration of 80mg/kg. Brain slices were taken after 3 days according to the processdescribed in Example 5. hC10-2_N32S specifically labels targetstructures in vivo in hippocampus and cortex in rTg4510 brains, but notin control tTA brains. Paired images for AlexaFluor488 and Hoechstsignal are shown in hippocampal sections.

FIG. 6 (Panels A-F). Decoration of tau structures in rTg4510 micefollowing i.v. injection of hC10-2_N32S_A101T (Panles A, C and Erepresent rTg4510; Panels B, D and F represent tTA). The mice received asingle injection of a volume of 150 μL of hC10-2_N32S_A101T antibodiesat a concentration of 80 mg/kg. Brain slices were taken after 3 daysaccording to the process described in Example 5. hC10-2_N32S_A101Tspecifically labels target structures in vivo in hippocampus and cortexin rTg4510 brains, but not in control tTA brains. Paired images forAlexaFluor488 and Hoechst signal are shown in hippocampal sections.

Comparing FIGS. 4-6 indicates that hC10.2, hC10-2_N32S andhC10-2_N32S_A101T cross the blood brain barrier upon intravenousinjection. The Figures further indicate that hC10-2_N32S andhC10-2_N32S_A101T labels tau structures (immunoreactive to tau tangles)the hippocampus and cortex with improved results compared to hC10-2.

FIG. 7 (Panels A-D). Tau species recognized by pS396 specific antibodiesin Alzheimer's diseased (AD) brain. As described in Example 6, insections of AD brain, tau tangles were either co-labelled by E1 and p396antibodies or positive for pS396 antibodies alone (arrows). The sectionswere analyzed by fluorescent microscopy. A number of tau tangles wereonly labelled by either hC10-2 and hC10-2_N32S_A101T antibodies(arrows). Given ghost tangles are not stained by N-terminal tauantibodies, the tau species labelled by hC10-2 or hC10-2_N32S_A101Tantibodies alone likely represent extracellular ghost tangles.

FIGS. 8A-8C. Detection of pathological tau by Western blot. As describedin Example 6 section “Detection of pathological tau by Western blot”,pathological tau with hC10.2, hC10-2_N32S, hC10-2_N32S_A101T by detectedby Western blot. Forebrain pooled from three rTg4510 mice andnon-transgenic (non-tg) control littermates euthanized at 32 weeks ofage and pooled cortical specimen from four AD mice and four healthycontrol (HC) donors, respectively were fractioned into soluble (S1),TBS-soluble pellet (Sip) and sarkosyl-insoluble (P3) fractions andanalyzed by western blot for phosphorylated tau at pS396 epitope with 1μg/ml hC10.2 (A), hC10-2_N32S (B), hC10-2_N32S_A101T (C). In rTg4510,normal human 4R0N tau is displayed at 55 kDa, while hyperphosphorylatedtau species are displayed at 64 kDa and 70 kDa. In AD,hyperphosphorylated tau species are displayed as four bands of 54, 64,69 kDa, and 74 kDa, with a variable amount of AD typical smear.

Each of hC10-2, hC10-2_N32S and hC10-2_N32S_A101T are selective for tauproteins of rTg4510 mice over non-transgenic mice and for AD donors overhealthy control donors. Moreover, in soluble (S1), TBS-soluble pellet(Sip) and sarkosyl-insoluble (P3) fractions, each of hC10-2, hC10-2_N32Sand hC10-2_N32S_A101T are selective to the pathogenic tau 64 kDa proteinof rTg4510 mice over the normal tau 55 kDa protein of rTg4510 mice.

FIG. 9. Immunoprecipitation of tau from AD brains. As described in theExample 6 section “Immunoprecipitation of pathological tau”immunoprecipitation of tau with 10 μg hC10.2, hC10-2_N32S,hC10-2_N32S_A101T, using human IgG1 control (hIgG1) from 500 μgpre-cleared lysates of cortical brain homogenates pooled from four ADand health control (HC) donors was analyzed by western blot withpolyclonal rabbit anti-pS396 tau (pS396 tau) antibody. In AD,hyperphosphorylated tau species are displayed as four bands of 54, 64,69 kDa, and 74 kDa, with a variable amount of AD typical smear.

FIGS. 10A-10C Quantification of tau aggregation by Cisbio assay. Wildtype (Wt) seeding material (WW) showed no seeding and background signalwas subtracted from all seeded samples. Tg4510 homogenates seededefficiently and the seeding effect was not affected by treatment withB12, but was effected to various degree by treatment in seeding studiesperformed at a concentration of 20 μg/mL with tau antibodies of theinvention (hC10-2_N32S_A101T>hC10-2_N32S>hC10-2). Graphs representresults of four independent sets of experiments and are plotted asrelative tau aggregation (fold signal over background normalized tototal protein) and relative insoluble p396 tau was quantified bydensitometry of western blots of the triton-X insoluble fraction (foldsignal over background normalized). All samples were normalized toisotype control antibody B12. FIG. 10 B-C presents a quantification oftau aggregation by Cisbio assay. Seeded pcDNA HEK293 cells showed nosignal, confirming the absence of detection for input seeding material.Wt (wild type) seeding material (WW) showed no seeding, but in contrastrTg4510 homogenates (CC) seeded efficiently, compared to unseeded. Thisseeding effect was not affected by treatment with HEL, but was partiallyreversed by treatment with tau antibodies (C10-2>D1.2>hACl36-2B6-Ab1).Graphs represent three independent sets of samples and are plotted asrelative tau aggregation (fold signal over background normalized tototal protein). Example 6, the section “Cell and Aggregation assay”,describes the protocol followed.

FIG. 11. Quantification of Tau5 western blot signal afterimmunodepletion of AD brain extracts using different amounts of hC10-2and 2.10.3 antibody. As described in Example 7, both antibodies didremove a small fraction of total tau from Alzheimer brain extracts.

FIG. 12. Quantification of P-S422 Tau western blot signal afterimmunodepletion AD brain extracts using different amounts of hC10-2(diamonds) and 2.10.3 (triangles) antibody. The figure shows resultsfrom Example 7. Tau phosphorylated at Serine 422 can be efficientlyremoved from AD brain extracts by immune depletion using either hC10-2or 2.10.3. Both antibodies did remove more than 90% P-S422 Tau, althoughmore of the 2.10.3 antibody was needed to reach the same effect.

FIG. 13. Quantification of pS396 Tau western blot signal afterimmunodepletion AD brain extracts using different amounts of hC10-2 and2.10.3 antibody. The figure shows results from Example 7. hC10-2immunodepletion removed 88% of tau phosphorylated at Serine 396, whereas2.10.3 only removed 55% of pS396 Tau from AD brain extracts.

FIG. 14. Quantification of P-S1991202 Tau western blot signal afterimmunodepletion of AD brain extracts using different amounts of hC10-2and 2.10.3 antibody. The figure shows results from Example 7. The hC10-2immunodepletion cleared 69% of tau being phosphorylated at Serine199/202. The 2.10.3 antibody did not give the same dose dependentreduction.

FIG. 15. Alzheimers diseased brain extracts on a western blot before andafter immune depletion. The figure shows results from Example 7. Thereis a 25 kDa Tau fragment phosphorylated at serine 396. Immunodepletionusing hC10-2 resulted in a reduction of the 25 kDa Tau band. The 2 otherphospho-specific antibodies 2.10.3 and AT8 did not remove this 25 kDaspecies.

FIG. 16. Quantification of pS396 Tau western blot signal afterimmunodepletion AD brain extracts using different amounts of hC10-2variants N32S, N32Q, N32S_A101T, N32Q_A101T, N32Q_D55E and N32S_D55E. Ascan be concluded from Example 8, the ability of the antibodies of theinvention to remove tau phosphorylated at Serine 396 from AD brainhomogenates was substantial. At less than 0.1 μg of antibody (data pointat 75 ng), the variants resulted in a decrease in the pS396 signal by atleast 28% (except for N32Q, D55E which was 16%) whereas the C10.2resulted in a decrease in the pS396 signal of less than 6%.

FIG. 17 (Panels A-D). Seeding of tau tangles the hippocampus, caused byinjecting AD brain extracts. As performed in Example 8a, at a dose of 15mg/kg, the mC10-2 treatment significantly reduced tangle pathology inthe seeded hippocampus by 57% (P<0,05). There was a clear trendindicating hC10-2 also reduced pathology. By comparison, 2.10.3 failedto show an effect, at the same dose.

FIG. 18. Residue P-Ser396 and Tyr394 are at the center of the antigenbinding site

The structure ofIle(392)-Val(393)-Tyr(394)-Lys(395)-pSer(396)-Pro(397)-Val(398) isshown. The main interaction with the antibody of the invention involvesthe hydrophobic pocket, the pSer396 and Y394 of tau peptide. There is anextensive hydrogen bonding network formed and charge/polar interactionsbetween the Y(394) sidechain and the backbone with phosphonate ofpSer396. The HC CDR1 of the antibodies of invention comprise thepalindromic 8-residue motif POLAR AA-HYRDROPHOBIC AA-POLAR AA-CHARGEDAA-CHARGED AA-POLAR AA-HYRDROPHOBIC AA-POLAR AA(Thr-Phe-Thr-Asp-Arg-Thr-Ile-His). The charged residues interact via anextensive bonding network formed by hydrogen bonding, charge/charge, andcharge/polar interactions between the antibody and the tau protein.

FIG. 19. Antibody efficacy in treatment paradigm

As performed in Example 8b, HEK293 cells expressing hTau-P301L wereseeded with Tg4510 homogenates pre-incubated with indicated antibodies,trypsinized and re-seeded 24 hours post (antibodies re-added) andharvested 48 h post-seeding. Total cell homogenates were probed foraggregated tau using Cisbio tau aggregation. Data are the pooled data of4 independent biological replicas +/−S.E.M., normalized to CC+B12(tg4510).

P301L-htau expressing cells were seeded with 40 μg Tg4510 brainhomogenate (total protein) pre-incubated o/n at 4° C. with antibodies(20 μg/ml≈133 nM) per 6-well. Seeding with CC+B12 gave a large seedingresponse. hC10.2 had an impact on aggregation approximately 40% Allother antibodies showed an at least comparable effect to hC10.2. Inparticular, the N32S and N32S_A101T variants of hC10.2 showed strongereffects 45% and 62% reduction in aggregated tau. The N32S_A101T variantshowed a significant stronger effect on aggregation compared to hC10.2.The Figure demonstrates that humanized C10.2 is effective at engagingtau induced seeding, but the addition of the N32S and in particular ofthe N32S and A101T double mutation increases the neutralizing activityof the mAb.

FIG. 20. Deamidation studies of the variants at stressed conditions Asperformed in Example 8c, deamidation of Asn residues at position 32 or34 of the VL chain was monitored by analyzing the tryptic peptide LC:T2[VTMTCQASQDTSIXLNWFQQKPGK; SEQ ID NO:58] by LC-MS. X is either Asn, Glnor Ser in the respective variants indicated in FIG. 20. The MS analysisallows the calculation of relative content of deamidated to nondeamidated peptide. In the WT, A101T and D55E variants extensivedeamidation at the LC:T2 peptide is observed. It is also clear thatchanging Asn32 to either Gln or Ser completely prevents deamidation ofthe peptide at the other Asn34 residues. Also, we do not detect anydeamidation of the Gln32 variants. Similar results were observed withvariants of the Asn34.

FIGS. 21A-21B. Reduction of tau seeding and aggregation in corticalneurons by tau antibodies

Tau seeding and aggregation in cortical neuronal cultures from rTg4510mouse embryos was induced by 0.2 ng pathological tau from P3 or S1pfractions from 40 weeks old rTg4510 mice and measured by the Cisbio tauaggregation assay. At 7 days in culture (DIV) neurons were treated witha mixture of P3 or S1p and 10 μg antibody or phosphate-buffered saline(PBS). Complete medium change was performed at DIV11 to remove residualP3 and S1p seeds and antibodies. Tau seeding was allowed for additional4 days and neurons lysed at DIV15 to measure tau seeding andaggregation. PBS and human control IgG antibody (IgG con ab) did notaffected tau seeding and aggregation. Tau seeding and aggregation waspartially reversed by treatment with tau antibodies(hC10.2_A101T_N32S>hC10.2_N32S>hC10.2). Following reduction of tauseeding and aggregation was measured: 23% with hC10.2, 41-53% withhC10.2_N32S, and 48-60% with hC10.2_A101T_N32S. The bar graphs representdata from two independent experiments of tau aggregation normalized toneuronal protein as means±SD. One way ANOVA Newman-Keuls MultipleComparison Test (PBS/IgG con ab vs hC10.2, hC10.2_N32S,hC10.2_A101T_N32S***p<0.001; hC10.2 vs hC10.2_N32S, hC10.2_A101T_N32S###p<0.001).

FIG. 22 (Panels A-D). Dose-dependent decoration of tau structures inrTg4510 mice following i.v. injection of hC10-2_N32S.

hC10-2_N32S specifically labels target structures in vivo in hippocampusand cortex in rTg4510 brains. Images from cingulate cortex are shown.Strongest signals at 20 and 80 mg/kg, weak signals at 8 mg/kg, no signalvisible at 0.8 mg/kg.

FIG. 23 Seeded tau pathology in hippocampus

The number of cells having Gallyas tangle staining in the seededhippocampus was reduced by hC10-2 (50%), hC10-2_N32S (48%) andhC10-2_N32S_A101T (47%) treatment. The quantification was made in every6^(th) sections covering the dorsal hippocampus, in total 8 sectionswere used per animal. The cell number reflects the sum of positive cellsin all sub-regions of the hippocampus identified in the 8 sections. Oneway anova and Dunnett's multiple comparisons test was used to analysethe data.

SEQUENCES INCORPORATED BY REFERENCE SEQ ID NO:1 Human tau (2N4R)

SEQ ID NO:2 tau residues 386-408 (pS396, pS404)

SEQ ID NO:3 C10-2 Light Chain CDR1 SEQ ID NO:4 C10-2 Light Chain CDR2SEQ ID NO:5 C10-2 Light Chain CDR3 SEQ ID NO:6 C10-2 Heavy Chain CDR1SEQ ID NO:7 C10-2 Heavy Chain CDR2 SEQ ID NO:8 C10-2 Heavy Chain CDR3SEQ ID NO:9 Mouse C10-2 Light Chain SEQ ID NO:10 Mouse C10-2 Heavy Chain

SEQ ID NO:11 humanized C10-2 Heavy ChainSEQ ID NO:12 humanized C10-2 Light ChainSEQ ID NO:13 humanized C10-2 Heavy Chain Variant D55ESEQ ID NO:14 humanized C10-2 Heavy Chain Variant D55QSEQ ID NO:15 humanized C10-2 Heavy Chain Variant D55SSEQ ID NO:16 humanized C10-2 Light Chain Variant N32SSEQ ID NO:17 humanized C10-2 Light Chain Variant N32QSEQ ID NO:18 humanized C10-2 Light Chain Variant N34SSEQ ID NO:19 humanized C10-2 Light Chain Variant N34QSEQ ID NO:20 humanized C10-2 Light Chain Variant N32S, N34SSEQ ID NO:21 humanized C10-2 Light Chain Variant N32Q, N34SSEQ ID NO:22 humanized C10-2 Light Chain Variant N32Q, N34QSEQ ID NO:23 humanized C10-2 Light Chain Variant N32S, N34QSEQ ID NO:24 humanized C10-2 Heavy Chain Variant A101TSEQ ID NO:25 humanized C10-2 Heavy Chain Variant D55E, A101TSEQ ID NO:26 humanized C10-2 Heavy Chain Variant D55Q, A101TSEQ ID NO:27 humanized C10-2 Heavy Chain Variant D55S, A101TSEQ ID NO:28 humanized C10-2 Heavy Chain CDR2 Variant D55ESEQ ID NO:29 humanized C10-2 Heavy Chain CDR2 Variant D55QSEQ ID NO:30 humanized C10-2 Heavy Chain CDR2 Variant D55SSEQ ID NO:31 humanized C10-2 Light Chain CDR1 Variant N32SSEQ ID NO:32 humanized C10-2 Light Chain CDR1 Variant N32QSEQ ID NO:33 humanized C10-2 Light Chain CDR1 Variant N34SSEQ ID NO:34 humanized C10-2 Light Chain CDR1 Variant N34QSEQ ID NO:35 humanized C10-2 Light Chain CDR1 Variant N32S, N34SSEQ ID NO:36 humanized C10-2 Light Chain CDR1 Variant N32Q, N34SSEQ ID NO:37 humanized C10-2 Light Chain CDR1 Variant N32Q, N34QSEQ ID NO:38 humanized C10-2 Light Chain CDR1 Variant N32S, N34QSEQ ID NO:39 humanized C10-2 Heavy Chain CDR3 Variant A101TSEQ ID NO:40 IMGT numbering humanized C10-2 Light Chain CDR1SEQ ID NO:41 IMGT numbering humanized C10-2 Light Chain CDR2SEQ ID NO:42 IMGT numbering humanized C10-2 Light Chain CDR3SEQ ID NO:43 IMGT numbering humanized C10-2 Heavy Chain CDR1SEQ ID NO:44 IMGT numbering humanized C10-2 Heavy Chain CDR2SEQ ID NO:45 IMGT numbering humanized C10-2 Heavy Chain CDR3SEQ ID NO:46 IMGT numbering humanized C10-2 Light Chain CDR1 VariantN32SSEQ ID NO:47 IMGT numbering humanized C10-2 Light Chain CDR2 VariantN32SSEQ ID NO:48 IMGT numbering humanized C10-2 Light Chain CDR3 VariantN32SSEQ ID NO:49 IMGT numbering humanized C10-2 Heavy Chain CDR1 VariantA101TSEQ ID NO:50 IMGT numbering humanized C10-2 Heavy Chain CDR2 VariantA101TSEQ ID NO:51 IMGT numbering humanized C10-2 Heavy Chain CDR3 VariantA101TSEQ ID NO:52 Chotia numbering humanized C10-2 Heavy Chain CDR1SEQ ID NO:53 Chotia numbering humanized C10-2 Heavy Chain CDR2SEQ ID NO:54 Chotia numbering humanized C10-2 Heavy Chain CDR3SEQ ID NO:55 Chotia numbering humanized C10-2 Heavy Chain CDR1 VariantA101TSEQ ID NO:56 Chotia numbering humanized C10-2 Heavy Chain CDR2 VariantA101TSEQ ID NO:57 Chotia numbering humanized C10-2 Heavy Chain CDR3 VariantA101TSEQ ID NO:58 5-residue motif of the HC CDR1SEQ ID NO:59 8-residue motif of the HC CDR1SEQ ID NO:60 first primer for the tTA activator transgeneSEQ ID NO:61 second primer for the tTA activator transgeneSEQ ID NO:62 first primer for the mutant tau responder transgeneSEQ ID NO:63 second primer for the mutant tau responder transgene

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “tau” is synonymous with “the tau protein” andrefers to any of the tau protein isoforms (identified in, for example,UniProt as P10636, 1-9). The amino acid numbering of tau that is usedherein is given with respect to isoform 2 (SEQ ID NO:1) as shown below,with methionine (M) being amino acid residue 1:

SEQ ID NO: 1: MAEPRQEFEV MEDHAGTYGL GDRKDQGGYT MHQDQEGDTD AGLKESPLQT PTEDGSEEPG SETSDAKSTP TAEDVTAPLV DEGAPGKQAA AQPHTEIPEG TTAEEAGIGD TPSLEDEAAG HVTQARMVSK SKDGTGSDDK KAKGADGKTK IATPRGAAPP GQKGQANATR IPAKTPPAPK TPPSSGEPPK SGDRSGYSSPGSPGTPGSRS RTPSLPTPPT REPKKVAVVR TPPKSPSSAK SRLQTAPVPM PDLKNVKSKI GSTENLKHQP GGGKVQIINK KLDLSNVQSK CGSKDNIKHV PGGGSVQIVY KPVDLSKVTS KCGSLGNIHH KPGGGQVEVK SEKLDFKDRV QSKIGSLDNI THVPGGGNKK IETHKLTFRE NAKAKTDHGA EIVYKSPVVSGDTSPRHLSN VSSTGSIDMV DSPQLATLAD EVSASLAKQG L

The present invention relates to antibodies and epitope-bindingfragments thereof that are capable of specifically binding to tau, andin particular to human tau, and in one embodiment exhibit the ability tospecifically bind to the phosphorylated S396 residue (pS396) of humantau. The antibodies and epitope-binding fragments thereof of theinvention, are further characterized by being incapable or substantiallyincapable of specifically binding to the phosphorylated serine 404(pS404) residue on human tau, for example under antibody limited ornon-saturating conditions. Furthermore, phosphorylation at pS404 doesnot interfere with the specific binding to pS396 containing epitopes. Asused herein, the notations “pS” and “^({p})S” denote the amino acidresidue phosphoserine and the subsequent numbers identify the positionof the residue relative to the sequence of SEQ ID NO:1. As used herein,an antibody is “substantially” incapable of binding to an epitope ifrelative to another epitope such binding is less than 20%, less than10%, less than 5%, less than 2%, and more preferably, less than 1% ofthe binding observed with such other epitope.

The term “antibody” (Ab) in the context of the present invention refersto an immunoglobulin molecule or according to some embodiments of theinvention, a fragment of an immunoglobulin molecule which has theability to specifically bind to an epitope of a molecule (“antigen”).Naturally occurring antibodies typically comprise a tetramer which isusually composed of at least two heavy chains (HC) and at least twolight chains (LC). Each heavy chain is comprised of a heavy chainvariable domain (abbreviated herein as VH) and a heavy chain constantdomain, usually comprised of three domains (CH1, CH2 and CH3). Humanheavy chains can be of any isotype, including IgG (IgG1, IgG2, IgG3 andIgG4 subtypes). Each light chain is comprised of a light chain variabledomain (abbreviated herein as VL) and a light chain constant domain(CL). Human light chains include kappa chains and lambda chains. Theheavy and light chain variable domain is typically responsible forantigen recognition, while the heavy and light chain constant domain maymediate the binding of the immunoglobulin to host tissues or factors,including various cells of the immune system (e.g., effector cells) andthe first component (C1q) of the classical complement system. The VH andVL domains can be further subdivided into domains of hypervariability,termed “complementarity determining regions,” that are interspersed withdomains of more conserved sequence, termed “framework regions” (FR).Each VH and VL is composed of three CDR Domains and four FR Domainsarranged from amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable domains of the heavy andlight chains contain a binding domain that interacts with an antigen. Ofparticular relevance are antibodies and their epitope-binding fragmentsthat have been “isolated” so as to exist in a physical milieu distinctfrom that in which it may occur in nature or that have been modified soas to differ from a naturally occurring antibody or theirepitope-binding fragments in amino acid sequence.

The term “epitope” means an antigenic determinant capable of specificbinding to an antibody. Epitopes usually consist of surface groupings ofmolecules such as amino acids or sugar side chains and usually havespecific three dimensional structural characteristics, as well asspecific charge characteristics. Conformational and linear epitopes aredistinguished in that the binding to the former, but not the latter, isalways lost in the presence of denaturing solvents. The epitope maycomprise amino acid residues directly involved in the binding and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically epitope-binding peptide (in other words, the amino acidresidue is within the footprint of the specifically epitope-bindingpeptide).

As used herein, the term “epitope-binding fragment of an antibody” meansa fragment, portion, region or domain of an antibody (regardless of howit is produced (e.g., via cleavage, recombinantly, synthetically, etc.)that is capable of specifically binding to an epitope. Anepitope-binding fragment may contain any 1, 2, 3, 4, 5 or all 6 of theCDR Domains of such antibody and, although capable of specificallybinding to such epitope, may exhibit a specificity, affinity orselectivity toward such epitope that differs from that of such antibody.Preferably, however, an epitope-binding fragment will contain all 6 ofthe CDR Domains of such antibody. An epitope-binding fragment of anantibody may be part of, or comprise, a single polypeptide chain (e.g.,an scFv), or may be part of, or comprise, two or more polypeptidechains, each having an amino-terminus and a carboxyl terminus (e.g., adiabody, a Fab fragment, a Fab₂ fragment, etc.). Fragments of antibodiesthat exhibit epitope-binding ability can be obtained, for example, byprotease cleavage of intact antibodies. More preferably, although thetwo domains of the Fv fragment, VL and VH, are naturally encoded byseparate genes, or polynucleotides that encode such gene sequences(e.g., their encoding cDNA) can be joined, using recombinant methods, bya flexible linker that enables them to be made as a single protein chainin which the VL and VH regions associate to form monovalentepitope-binding molecules (known as single-chain Fv (scFv); see e.g.,Bird et al., (1988) Science 242:423-426; and Huston et al. (1988) Proc.Natl. Acad. Sci. (U.S.A.) 85:5879-5883). Alternatively, by employing aflexible linker that is too short (e.g., less than about 9 residues) toenable the VL and VH domains of a single polypeptide chain to associatetogether, one can form a bispecific antibody, diabody, or similarmolecule (in which two such polypeptide chains associate together toform a bivalent epitope-binding molecule) (see for instance PNAS USA90(14), 6444-8 (1993) for a description of diabodies). Examples ofepitope-binding fragments encompassed within the present inventioninclude (i) a Fab′ or Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains, or a monovalent antibody as described inWO2007059782; (ii) F(ab′)2 fragments, bivalent fragments comprising twoFab fragments linked by a disulfide bridge at the hinge domain; (iii) anFd fragment consisting essentially of the VH and CH1 domains; (iv) a Fvfragment consisting essentially of a VL and VH domains, (v) a dAbfragment (Ward et al., Nature 341, 544-546 (1989)), which consistsessentially of a VH domain and also called domain antibodies (Holt etal; Trends Biotechnol. 2003 November; 2i(II):484-90); (vi) camelid ornanobodies (Revets et al; Expert Opin Biol Ther. 2005 January; 5 (I):III-24) and (vii) an isolated complementarity determining region (CDR).Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they may be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH domains pair to form monovalentmolecules (known as single chain antibodies or single chain Fv (scFv),see for instance Bird et al., Science 242, 423-426 (1988) and Huston etal., PNAS USA 85, 5879-5883 (1988)). These and other useful antibodyfragments in the context of the present invention are discussed furtherherein. It also should be understood that the term antibody, unlessspecified otherwise, also includes antibody-like polypeptides, such aschimeric antibodies and humanized antibodies, and antibody fragmentsretaining the ability to specifically bind to the antigen(epitope-binding fragments) provided by any known technique, such asenzymatic cleavage, peptide synthesis, and recombinant techniques. Anantibody as generated can possess any isotype. As used herein, “isotype”refers to the immunoglobulin class (for instance IgG1, IgG2, IgG3 orIgG4) that is encoded by heavy chain constant domain genes. Suchantibody fragments are obtained using conventional techniques known tothose of skill in the art; suitable fragments capable of binding to adesired epitope may be readily screened for utility in the same manneras an intact antibody. In one embodiment, the Fc region of theantibodies of the invention comprise a mutation that modulates effectorfunctions.

The term “bispecific antibody” refers to an antibody containing twoindependent epitope-binding fragments that each target independenttargets. These targets can be epitopes present on different proteins ordifferent epitopes present on the same target. Bispecific antibodymolecules can be made using compensatory amino acid changes in theconstant domains of the HCs of the parent monospecific bivalent antibodymolecules. The resulting heterodimeric antibody contains one Fabscontributed from two different parent monospecific antibodies. Aminoacid changes in the Fc domain leads to increased stability of theheterodimeric antibody with bispecificity that is stable over time.(Ridgway et al., Protein Engineering 9, 617-621 (1996), Gunasekaran etal., JBC 285, 19637-1(2010), Moore et al., MAbs 3:6 546-557 (2011),Strop et al., JMB 420, 204-219 (2012), Metz et al., Protein Engineering25:10 571-580 (2012), Labrijn et al., PNAS 110:113, 5145-5150 (2013),Spreter Von Kreudenstein et al., MAbs 5:5 646-654 (2013)). Bispecificantibodies can also include molecules that are generated using ScFvfusions. Two monospecific scfv are then independently joined to Fcdomains able to form stable heterodimers to generate a single bispecificmolecule (Mabry et al., PEDS 23:3 115-127 (2010). Bispecific moleculeshave dual binding capabilities.

The terms “C10-2”, “human C10-2”, “hC10-2”, “HC10-2”, “hC10.2”,“Humanized C10-2” and “humanized C10-2” as used herein and in theFigures are intended to be synonymous and are defined as Antibody C10-2.The term is intended to denote an antibody or an epitope-bindingfragment thereof comprising, or consisting of, an antibody Light ChainVariable domain having:

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and an antibody Heavy Chain Variable domain having:(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Antibody C10-2 is a humanized antibody which may be defined ascomprising the heavy chain of SEQ ID NO:11, the light chain of SEQ IDNO:12., or both. One embodiment of the invention is directed to anantibody or epitope-binding fragment thereof comprising of the heavychain of SEQ ID NO:11 and the light chain of SEQ ID NO:12.

The term “mC10-2” as used herein and in the Figures is intended to meanmouse antibody C10-2 and is defined by SEQ ID. NO. 9 and 10. Mouseantibody C10.2 is used as a control antibody and is not part of theinvention.

The terms “hC10-2_N32S” and “C10-2_N32S” as used herein and in theFigures are intended to be synonymous and are variants of Antibody C10-2wherein the light chain has been mutated to at least comprise mutationof amino acid residue 32 from N to S and is defined as Antibody N32S.The terms “hC10-2_N32Q” and “C10-2_N32Q” as used herein and in theFigures are intended to be synonymous and are variants of Antibody C10-2wherein the light chain has been mutated to at least comprise mutationof amino acid residue 32 from N to Q and is defined as Antibody N32Q.

The terms “hC10-2_N34S” and “C10-2_N34S” as used herein and in theFigures are intended to be synonymous and are variants of Antibody C10-2wherein the light chain has been mutated to at least comprise mutationof amino acid residue 34 from N to S and is defined as Antibody N34S.The terms “hC10-2_N34Q” and “C10-2_N34Q” as used herein and in theFigures are intended to be synonymous and are variants of Antibody C10-2wherein the light chain has been mutated to at least comprise mutationof amino acid residue 34 from N to Q and is defined as Antibody N34Q.

The terms “hC10-2_N32S_N34S” and “C10-2_N32S_N34S” as used herein and inthe Figures are intended to be synonymous and are variants of AntibodyC10-2 wherein the light chain has been mutated to at least comprisemutations of amino acid residues 32 and 34 from N to S and is defined asAntibody N32S, N34S. The terms “hC10-2_N32Q_N34S” and “C10-2_N32Q_N34S”as used herein and in the Figures are intended to be synonymous and arevariants of Antibody C10-2 wherein the light chain has been mutated toat least comprise mutations of amino acid residues 32 and 34 from N to Qand to N to S, respectively and is defined as Antibody N32Q, N34S. Theterms “hC10-2_N32Q_N34Q” and “C10-2_N32Q_N34Q” as used herein and in theFigures are intended to be synonymous and are variants of Antibody C10-2wherein the light chain has been mutated to at least comprise mutationsof amino acid residues 32 and 34 from N to Q and is defined as AntibodyN32Q, N34Q. The terms “hC10-2_N32S_N34Q” and “C10-2_N32S_N34Q” as usedherein and in the Figures are intended to be synonymous and are variantsof Antibody C10-2 wherein the light chain has been mutated to at leastcomprise mutations of amino acid residues 32 and 34 from N to S and to Nto Q, respectively and is defined as Antibody N32S, N34Q.

The terms “hC10-2_D55E” and “C10-2_D55E” as used herein and in theFigures are intended to be synonymous and are variants of Antibody C10-2wherein the heavy chain has been mutated to at least comprise mutationof amino acid residue 55 from D to E and is defined as Antibody D55E.The term “hC10-2_D55Q”, “C10-2_D55Q” as used herein and in the Figuresare intended to be synonymous and are variants of Antibody C10-2 whereinthe heavy chain has been mutated to at least comprise mutation of aminoacid residue 55 from D to Q and is defined as Antibody D55Q. The term“hC10-2_D55S”, “C10-2 D55S” as used herein and in the Figures areintended to be synonymous and are variants of Antibody C10-2 wherein theheavy chain has been mutated to at least comprise mutation of amino acidresidue 55 from D to S and is defined as Antibody D55S.

The terms “hC10-2_A101T” and “C10-2_A101T” as used herein and in theFigures are intended to be synonymous and are variants of Antibody C10-2wherein the heavy chain has been mutated to at least comprise mutationof amino acid residue 101 from A to T and is defined as Antibody A101T.

The term “hC10-2_N32S_A101T”, “C10-2_N32S_A101T”, hC10-2_A101T_N32S″ and“C10-2_A101T_N32S as used herein and in in the Figures are intended tobe synonymous and are variants of Antibody C10-2 wherein the heavy chainhas been mutated to at least comprise mutation of amino acid residue 101from A to T and wherein the light chain has been mutated to at leastcomprise mutation of amino acid residue 32 from N to S and is defined asAntibody N32S, A101T.

The term “hC10-2_N32Q_A101T”, “C10-2_N32Q_A101T”, hC10-2_A101T_N32Q” and“C10-2_A101T_N32Q as used herein and in in the Figures are intended tobe synonymous and are variants of Antibody C10-2 wherein the heavy chainhas been mutated to at least comprise mutation of amino acid residue 101from A to T and wherein the light chain has been mutated to at leastcomprise mutation of amino acid residue 32 from N to Q and is defined asAntibody N32Q, A101T.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A conventional monoclonal antibody compositiondisplays a single binding specificity and affinity for a particularepitope. In certain embodiments, a monoclonal antibody can be composedof more than one Fab domain thereby increasing the specificity to morethan one target. The terms “monoclonal antibody” or “monoclonal antibodycomposition” are not intended to be limited by any particular method ofproduction (e.g., recombinant, transgenic, hybridoma, etc.).

The antibodies of the present invention and epitope-binding fragmentsthereof will preferably be “humanised,” particularly if employed fortherapeutic purposes. The term “humanised” refer to a molecule,generally prepared using recombinant techniques, having anepitope-binding site derived from an immunoglobulin from a non-humanspecies and a remaining immunoglobulin structure based upon thestructure and/or sequence of a human immunoglobulin. The epitope-bindingsite may comprise either complete non-human antibody variable domainsfused to human constant domains, or only the complementarity determiningregions (CDRs), or parts hereof, of such variable domains grafted toappropriate human framework regions of human variable domains. Theframework residues of such humanized molecules may be wild type (e.g.,fully human) or they may be modified to contain one or more amino acidsubstitutions not found in the human antibody whose sequence has servedas the basis for humanization. Humanisation lessens or eliminates thelikelihood that a constant domain of the molecule will act as animmunogen in human individuals, but the possibility of an immuneresponse to the foreign variable domain remains (LoBuglio, A. F. et al.(1989) “Mouse/Human Chimeric Monoclonal Antibody In Man: Kinetics AndImmune Response,” Proc. Natl. Acad. Sci. (U.S.A.) 86:4220-4224). Anotherapproach focuses not only on providing human-derived constant domains,but modifying the variable domains as well so as to reshape them asclosely as possible to human form. It is known that the variable domainsof both heavy and light chains contain three complementarity-determiningregions (CDRs) which vary in response to the antigens in question anddetermine binding capability, flanked by four framework regions (FRs)which are relatively conserved in a given species and which putativelyprovide scaffolding for the CDRs. When nonhuman antibodies are preparedwith respect to a particular antigen, the variable domains can be“reshaped” or “humanized” by grafting CDRs derived from nonhumanantibody on the FRs present in the human antibody to be modified.Application of this approach to various antibodies has been reported bySato, K. et al. (1993) Cancer Res 53:851-856. Riechmann, L. et al.(1988) “Reshaping Human Antibodies for Therapy”, Nature 332:323-327;Verhoeyen, M. et al. (1988) “Reshaping Human Antibodies: Grafting AnAntilysozyme Activity,” Science 239:1534-1536; Kettleborough, C. A. etal. (1991) “Humanization Of A Mouse Monoclonal Antibody By CDR-Grafting:The Importance Of Framework Residues On Loop Conformation,” ProteinEngineering 4:773-3783; Maeda, H. et al. (1991) “Construction OfReshaped Human Antibodies With HIV-Neutralizing Activity”, HumanAntibodies Hybridoma 2:124-134; Gorman, S. D. et al. (1991) “Reshaping ATherapeutic CD4 Antibody,” Proc. Natl. Acad. Sci. (U.S.A.) 88:4181-4185;Tempest, P. R. et al. (1991) “Reshaping A Human Monoclonal Antibody ToInhibit Human Respiratory Syncytial Virus Infection in vivo”,Bio/Technology 9:266-271; Co, M. S. et al. (1991) “Humanized AntibodiesFor Antiviral Therapy”, Proc. Natl. Acad. Sci. (U.S.A.) 88:2869-2873;Carter, P. et al. (1992) “Humanization Of An Anti-p185her2 Antibody ForHuman Cancer Therapy,” Proc. Natl. Acad. Sci. (U.S.A.) 89:4285-4289; andCo, M. S. et al. (1992) “Chimeric And Humanized Antibodies WithSpecificity For The CD33 Antigen,” J. Immunol. 148:1149-1154. In someembodiments, humanised antibodies preserve all CDR sequences (forexample, a humanized mouse antibody which contains all six CDRs from themouse antibodies). In other embodiments, humanised antibodies have oneor more CDRs (one, two, three, four, five, six) which are altered withrespect to the original antibody, which are also termed one or more CDRs“derived from” one or more CDRs from the original antibody. The abilityto humanise an antibody is well known (see, e.g., U.S. Pat. Nos.5,225,539; 5,530,101; 5,585,089; 5,859,205; 6,407,213; 6,881,557).

The term “antibody “XX” is intended to denote an antibody orepitope-binding fragment thereof (for example antibody “C10-2”),comprising or consisting of: (a) the Light Chain and either the HeavyChain, the Heavy Chain Variable Domain, or the Heavy Chain VariableDomain CDR1-3 as defined by its respective SEQ ID NO, or (b) the LightChain Variable domain and either the Heavy Chain, the Heavy ChainVariable Domain, or the Heavy Chain Variable Domain CDR1-3 as defined byits respective SEQ ID NO, or (c) the Light Chain Variable domain CDR1-3,as defined by its respective SEQ ID NO, and either the Heavy Chain, theHeavy Chain Variable Domain, or the Heavy Chain Variable Domain CDR1-3as defined by its respective SEQ ID NO. In certain embodiments, theantibody or epitope-binding fragment thereof are defined by their entireHeavy Chain Variable Domain comprising as defined by their SEQ ID NO andtheir Light Chain Variable Domain as defined by their SEQ ID NO.

Unless otherwise specified herein, numbering of amino acid residues inthe Fc region or constant domain of an antibody is according to the EUnumbering system, also called the EU index, as described in Kabat etal., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991.

In some antibodies, only part of a CDR, namely the subset of CDRresidues required for binding, termed the SDRs, are needed to retainbinding in a humanized antibody. CDR residues not contacting therelevant epitope and not in the SDRs can be identified based on previousstudies. For example, residues Tyr Ser Gln Lys Phe Gln, corresponding toresidues 60-65 of the HC of SEQ ID NO. 11 are often not required fromregions of Kabat CDRs (these are also found in HC CDR2 (SEQ.ID. NO. 7),lying outside Chothia hypervariable loops (see, Kabat et al. (1992)SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, National Institutes ofHealth Publication No. 91-3242; Chothia, C. et al. (1987) “CanonicalStructures For The Hypervariable Regions Of Immunoglobulins,” J. Mol.Biol. 196:901-917), by molecular modeling and/or empirically, or asdescribed in Gonzales, N. R. et al. (2004) “SDR Grafting Of A MurineAntibody Using Multiple Human Germline Templates To Minimize ItsImmunogenicity,” Mol. Immunol. 41:863-872. In such humanized antibodiesat positions in which one or more donor CDR residues is absent or inwhich an entire donor CDR is omitted, the amino acid occupying theposition can be an amino acid occupying the corresponding position (byKabat numbering) in the acceptor antibody sequence. The number of suchsubstitutions of acceptor for donor amino acids in the CDRs to includereflects a balance of competing considerations. Such substitutions arepotentially advantageous in decreasing the number of mouse amino acidsin a humanized antibody and consequently decreasing potentialimmunogenicity. However, substitutions can also cause changes ofaffinity, and significant reductions in affinity are preferably avoided.Positions for substitution within CDRs and amino acids to substitute canalso be selected empirically.

Antibodies are also characterized according to the IMGT numberingsystem, which is well defined in the field. The length (in number ofamino acids, that is in number of occupied positions) is a crucial andoriginal concept of IMGT-ONTOLOGY (http://www.imgt.org). The CDR-IMGTlengths characterize the IG and TR V-REGIONs of the germline genes andthe V-DOMAINs of the rearranged genes, cDNAs and proteins.

Antibodies are may for example be characterized according to the ChothiaNumbering Scheme http://www.bioinf.org.uk/abs/. The Chothia numberingscheme is identical to the Kabat scheme, but places the insertions inCDR-L1 and CDR-H1 at the structurally defined positions. The Chothianumbering scheme is based on the location of the structural loopregions. This means that topologically equivalent residues in theseloops do get the same label (unlike the Kabat scheme).

The fact that a single amino acid alteration of a CDR residue can resultin loss of functional binding (Rudikoff, S. etc. (1982) “Single AminoAcid Substitution Altering Antigen-Binding Specificity,” Proc. Natl.Acad. Sci. (USA) 79(6):1979-1983) provides a means for systematicallyidentifying alternative functional CDR sequences. In one preferredmethod for obtaining such variant CDRs, a polynucleotide encoding theCDR is mutagenized (for example via random mutagenesis or by asite-directed method (e.g., polymerase chain-mediated amplification withprimers that encode the mutated locus)) to produce a CDR having asubstituted amino acid residue. By comparing the identity of therelevant residue in the original (functional) CDR sequence to theidentity of the substituted (non-functional) variant CDR sequence, theBLOSUM62.iij substitution score for that substitution can be identified.The BLOSUM system provides a matrix of amino acid substitutions createdby analyzing a database of sequences for trusted alignments (Eddy, S. R.(2004) “Where Did The BLOSUM62 Alignment Score Matrix Come From?,”Nature Biotech. 22(8):1035-1036; Henikoff, J. G. (1992) “Amino acidsubstitution matrices from protein blocks,” Proc. Natl. Acad. Sci. (USA)89:10915-10919; Karlin, S. et al. (1990) “Methods For Assessing TheStatistical Significance Of Molecular Sequence Features By Using GeneralScoring Schemes,” Proc. Natl. Acad. Sci. (USA) 87:2264-2268; Altschul,S. F. (1991) “Amino Acid Substitution Matrices From An InformationTheoretic Perspective,” J. Mol. Biol. 219, 555-565. Currently, the mostadvanced BLOSUM database is the BLOSUM62 database (BLOSUM62.iij). Table1 presents the BLOSUM62.iij substitution scores (the higher the scorethe more conservative the substitution and thus the more likely thesubstitution will not affect function). If an epitope-binding fragmentcomprising the resultant CDR fails to bind to tau, for example, then theBLOSUM62.iij substitution score is deemed to be insufficientlyconservative, and a new candidate substitution is selected and producedhaving a higher substitution score. Thus, for example, if the originalresidue was glutamate (E), and the non-functional substitute residue washistidine (H), then the BLOSUM62.iij substitution score will be 0, andmore conservative changes (such as to aspartate, asparagine, glutamine,or lysine) are preferred.

The invention thus contemplates the use of random mutagenesis toidentify improved CDRs. In the context of the present invention,conservative substitutions may be defined by substitutions within theclasses of amino acids reflected in one or more of Tables 2, 3, or 4:

Amino Acid Residue Classes for Conservative Substitutions:

TABLE 2 Acidic Residues Asp (D) and Glu (E) Basic Residues Lys (K), Arg(R), and His (H) Hydrophilic Uncharged Residues Ser (S), Thr (T), Asn(N), and Gln (Q) Aliphatic Uncharged Residues Cly (G), Ala (A), Val (V),Leu (L), and Ile (I) Non-polar Uncharged Residues Cys (C), Met (M), andPro (P) Aromatic Residues Phe (F), Tyr (Y), and Trp (W)

Alternative Conservative Amino Acid Residue Substitution Classes:

TABLE 3 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

Alternative Physical and Functional Classifications of Amino AcidResidues:

TABLE 4 Alcohol Group-Containing Residues S and T Aliphatic Residues I,L, V and M Cycloalkenyl-Associated Residues F, H, W and Y HydrophobicResidues A, C, F, G, H, I, L, M, R, T, V, W and Y Negatively ChargedResidues D and E Polar Residues C, D, E, H, K, N, Q, R, S and TPositively Charged Residues H, K and R Small Residues A, C, D, G, N, P,S, T and V Very Small Residues A, G and S Residues Involved In TurnFormation A, C, D, E, G, H, K, N, Q, R, S, P and T Flexible Residues Q,T, K, S, G, P, D, E and R

More conservative substitutions groupings include:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine.

Additional groups of amino acids may also be formulated using theprinciples described in, e.g., Creighton (1984) Proteins: Structure andMolecular Properties (2d Ed. 1993), W. H. Freeman and Company.

Phage display technology can alternatively be used to increase (ordecrease) CDR affinity. This technology, referred to as affinitymaturation, employs mutagenesis or “CDR walking” and re-selection usesthe target antigen or an antigenic epitope-binding fragment thereof toidentify antibodies having CDRs that bind with higher (or lower)affinity to the antigen when compared with the initial or parentalantibody (See, e.g. Glaser et al. (1992) J. Immunology 149:3903).Mutagenizing entire codons rather than single nucleotides results in asemi-randomized repertoire of amino acid mutations. Libraries can beconstructed consisting of a pool of variant clones each of which differsby a single amino acid alteration in a single CDR and which containvariants representing each possible amino acid substitution for each CDRresidue. Mutants with increased (or decreased) binding affinity for theantigen can be screened by contacting the immobilized mutants withlabeled antigen. Any screening method known in the art can be used toidentify mutant antibodies with increased or decreased affinity to theantigen (e.g., ELISA) (See Wu et al. 1998, Proc. Natl. Acad. Sci.(U.S.A.) 95:6037; Yelton et al., 1995, J. Immunology 155:1994). CDRwalking which randomizes the Light Chain may be used possible (see,Schier et al., 1996, J. Mol. Bio. 263:551).

Methods for accomplishing such affinity maturation are described forexample in: Krause, J. C. et al. (2011) “An Insertion Mutation ThatDistorts Antibody Binding Site Architecture Enhances Function Of A HumanAntibody,” MBio. 2(1) pii: e00345-10. doi: 10.1128/mBio.00345-10; Kuan,C. T. et al. (2010) “Affinity-Matured Anti-Glycoprotein NMB RecombinantImmunotoxins Targeting Malignant Gliomas And Melanomas,” Int. J. Cancer10.1002/ijc.25645; Hackel, B. J. et al. (2010) “Stability And CDRComposition Biases Enrich Binder Functionality Landscapes,” J. Mol.Biol. 401(1):84-96; Montgomery, D. L. et al. (2009) “Affinity MaturationAnd Characterization Of A Human Monoclonal Antibody Against HIV-1 gp41,”MAbs 1(5):462-474; Gustchina, E. et al. (2009) “Affinity Maturation ByTargeted Diversification Of The CDR-H2 Loop Of A Monoclonal Fab DerivedFrom A Synthetic Naïve Human Antibody Library And Directed Against TheInternal Trimeric Coiled-Coil Of Gp41 Yields A Set Of Fabs With ImprovedHIV-1 Neutralization Potency And Breadth,” Virology 393(1):112-119;Finlay, W. J. et al. (2009) “Affinity Maturation Of A Humanized RatAntibody For Anti-RAGE Therapy: Comprehensive Mutagenesis Reveals A HighLevel Of Mutational Plasticity Both Inside And Outside TheComplementarity-Determining Regions,” J. Mol. Biol. 388(3):541-558;Bostrom, J. et al. (2009) “Improving Antibody Binding Affinity AndSpecificity For Therapeutic Development,” Methods Mol. Biol.525:353-376; Steidl, S. et al. (2008) “In Vitro Affinity Maturation OfHuman GM-CSF Antibodies By Targeted CDR-Diversification,” Mol. Immunol.46(1):135-144; and Barderas, R. et al. (2008) “Affinity Maturation OfAntibodies Assisted By In Silico Modeling,” Proc. Natl. Acad. Sci. (USA)105(26):9029-9034.

Thus, the sequence of CDR variants of encompassed antibodies or theirepitope-binding fragments may differ from the sequence of the CDR of theparent antibody, 010-2 and C10.2 Variants, through substitutions; forinstance substituted 4 amino acid residue, 3 amino acid residue, 2 aminoacid residue or 1 of the amino acid residues. According to an embodimentof the invention it is furthermore envisaged that the amino acids in theCDR regions may be substituted with conservative substitutions, asdefined in the 3 tables above. For example, the acidic residue Asp canbe substituted with Glu without substantially affecting the bindingcharacteristic of the antibody.

As used herein, an antibody or an epitope-binding fragment thereof issaid to “specifically” bind a region of another molecule (i.e., anepitope) if it reacts or associates more frequently, more rapidly, withgreater duration and/or with greater affinity or avidity with thatepitope relative to alternative epitopes. It is also understood byreading this definition that, for example, an antibody orepitope-binding fragment thereof that specifically binds to a firsttarget may not or may not specifically or preferentially bind to asecond target. As used herein, the term “binding” in the context of thebinding of an antibody to a predetermined antigen typically refers tobinding with an affinity corresponding to a KD of about 10⁻⁷ M or less,such as about 10⁻⁸ M or less, such as about 10⁻⁹ M or less whendetermined by for instance surface plasmon resonance (SPR) technology ina BIAcore® 3000 instrument using the antigen as the ligand and theantibody as the analyte, and binds to the predetermined antigen with anaffinity corresponding to a KD that is at least ten-fold lower, such asat least 100 fold lower, for instance at least 1,000 fold lower, such asat least 10,000 fold lower, for instance at least 100,000 fold lowerthan its affinity for binding to a non-specific antigen (e.g., BSA,casein) other than the predetermined antigen or a closely-relatedantigen. The amount with which the affinity is lower is dependent on theKD of the antibody, so that when the KD of the antibody is very low(that is, the antibody is highly specific), then the amount with whichthe affinity for the antigen is lower than the affinity for anon-specific antigen may be at least 10,000 fold.

The term “kd” (sec−1 or 1/s), as used herein, refers to the dissociationrate constant of a particular antibody-antigen interaction. Said valueis also referred to as the koff value.

The term “ka” (M−1×sec−1 or 1/Msec), as used herein, refers to theassociation rate constant of a particular antibody-antigen interaction.

The term “KD” (M), as used herein, refers to the dissociationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the kd by the ka.

The term “KA” (M−1 or 1/M), as used herein, refers to the associationequilibrium constant of a particular antibody-antigen interaction and isobtained by dividing the ka by the kd.

In one embodiment, the invention relates to an anti-tau antibody, orepitope-binding fragment thereof, which exhibits one or more of thefollowing properties:

-   -   (i) a substantial inability to bind to non-phosphorylated tau;    -   (ii) a substantial inability to bind to tau that is        phosphorylated at S404 and not phosphorylated at S396;    -   (iii) the ability to bind to tau phosphorylated at S396;    -   (iv) the ability to bind to tau phosphorylated at both S396 and        at S404;    -   (v) the ability to selectively discriminate between        phosphorylated tau residues S396 and S404 such that it is        substantially unable to bind the phosphorylated 404 residue        (pS404);    -   (vi) the ability to bind hyper-phosphorylated tau from human        Alzheimer's disease brains;    -   (vii) the ability to discriminate between pathological and        non-pathological human tau protein; and/or    -   (viii) the capability, when used as described herein with        immune-depleted rTg4510 extracts from transgenic mice, to        specifically reduce the hyperphosphorylated tau 64 kDa and 70        kDa bands by at least 90%, while not reducing the 55 kDa tau        band by more than 10%; or the capability, when used as described        herein with extracts from human AD post-mortem brains to        specifically reduce the S396 phosphorylated hyperphosphorylated        tau bands by at least 90%, while not reducing the        non-hyperphosphorylated tau bands by more than 10%.

A further embodiment of the invention relates to a method for obtaininghigh specificity, high affinity antibodes, wherein the method comprisesthe steps of:

-   -   (A) injecting an immunogen into a mammal, to thereby immunize        said mammal;    -   (B) repeating said immunization of said mammal two or more        times;    -   (C) screening a serum sample from said repeatedly immunized        mammal for the presence of the desired high specificity, high        affinity antibodies but substantially less capable of binding to        other protein; and    -   (D) recovering said high specificity, high affinity antibodies.

The invention thus relates to a high specificity, high affinity antibodyspecific for pathogenic hyperphosphorylated tau comprising residue aphosphorylated S396. Such antibodies may be generated by adaptation ofthe above-indicated method for generating high specificity, highaffinity antibodies by:

-   -   (A) injecting an immunogen into a mammal, said immunogen        comprising the bi-phosphorylated peptide comprising 18-40, such        as at 18-30, such as 20-30 amino consecutive acid residues        comprising TDHGAEIVYK^({p})SPVVSGDT^({p})SPRHL (SEQ ID NO:2)        covering residues 386-410 of 2N4R tau, to thereby immunize said        mammal;    -   (B) repeating said immunization of said mammal two or more        times;    -   (C) screening a serum sample from said repeatedly immunized        mammal for the presence of high specificity, high affinity        antibodies capable of binding pathogenic hyperphosphorylated tau        comprising residue a phosphorylated S396, but substantially less        capable of binding non-pathogenic tau; and    -   (D) recovering said high specificity, high affinity antibodies.

As used herein, a “substantial inability” to bind a tau molecule denotesmore than a 20% difference, more than a 40% difference, more than a 60%difference, more than an 80% difference, more than a 100% difference,more than a 150% difference, more than a 2-fold difference, more than a4-fold difference, more than a 5-fold difference, or more than a 10-folddifference in functionality, relative to the detectable binding mediatedby a reference antibody.

The term “selective” and “immunoselective” when referring to the bindingcapabilities of an anti-tau antibody with respect to two epitopes isintended to denote that the observed binding under saturating conditionsexhibits at least an 80% difference, at least a 95% difference, and mostpreferably a 100% difference (i.e., no detectable binding to oneepitope). The term “selective” and “immunoselective” when referring to atau antibody is further intended to mean the antibody bindshyper-phosphorylated tau from human Alzheimer's disease brains and isable to discriminate between pathological and non-pathological human tauprotein.

The terms TBS-extractable (S1), high salt/sarkosyl-extractable (S3), andsarkosyl-insoluble (P3) fractions are fractions as obtained by the taubiochemical fractionation described herein.

The term “normal tau” refers to normal brain tau containing 2-3 moles ofphosphate per mole of the protein.

The term “hyperphosphorylated tau” refers to a poly-phosphorylatedspecies of tau consistent with poly-anionic species induced mobilityshift in Western Blot or to a tau species which has more than five, sixor seven Serine, Threonine or Tyrosine sites phosphorylated.

The term “tau having residue 396 phosphorylated” relates tohyperphosphorylated tau wherein the serine residue 396 isphosphorylated.

The term “transgenic non-human animal” refers to a non-human animalhaving a genome comprising one or more human heavy and/or light chaintransgenes or trans-chromosomes (either integrated or non-integratedinto the animal's natural genomic DNA) and which is capable ofexpressing fully human antibodies. For example, a transgenic mouse canhave a human light chain transgene and either a human heavy chaintransgene or human heavy chain trans-chromosome, such that the mouseproduces human anti-tau antibody when immunized with tau antigen and/orcells expressing tau. The human heavy chain transgene may be integratedinto the chromosomal DNA of the mouse, as is the case for transgenicmice, for instance HuMAb mice, such as HCo7 or HCol2 mice, or the humanheavy chain transgene may be maintained extra-chromosomally, as is thecase for trans-chromosomal KM mice as described in WO02/43478. Suchtransgenic and trans-chromosomal mice (collectively referred to hereinas “transgenic mice”) are capable of producing multiple isotypes ofhuman monoclonal antibodies to a given antigen (such as IgG, IgA, IgM,IgD and/or IgE) by undergoing V-D-J recombination and isotype switching.

Transgenic, nonhuman animal can also be used for production ofantibodies against a specific antigen by introducing genes encoding suchspecific antibody, for example by operatively linking the genes to agene which is expressed in the milk of the animal.

The term “treatment” or “treating” as used herein means ameliorating,slowing, attenuating, or reversing the progress or severity of a diseaseor disorder, or ameliorating, slowing, attenuating, or reversing one ormore symptoms or side effects of such disease or disorder. For purposesof this invention, “treatment” or “treating” further means an approachfor obtaining beneficial or desired clinical results, where “beneficialor desired clinical results” include, without limitation, alleviation ofa symptom, diminishment of the extent of a disorder or disease,stabilized (i.e., not worsening) disease or disorder state, delay orslowing of the progression a disease or disorder state, amelioration orpalliation of a disease or disorder state, and remission of a disease ordisorder, whether partial or total.

An “effective amount,” when applied to an antibody or epitope-bindingfragment thereof of the invention, refers to an amount sufficient, atdosages and for periods of time necessary, to achieve an intendedbiological effect or a desired therapeutic result including, withoutlimitation, clinical results. The phrase “therapeutically effectiveamount,” when applied to an antibody or an epitope-binding fragmentthereof of the invention, is intended to denote an amount of theantibody, or epitope-binding fragment thereof, that is sufficient toameliorate, palliate, stabilize, reverse, slow, attenuate or delay theprogression of a disorder or disease state, or of a symptom of thedisorder or disease. In an embodiment, the method of the presentinvention provides for administration of the antibody, orepitope-binding fragment thereof, in combinations with other compounds.In such instances, the “effective amount” is the amount of thecombination sufficient to cause the intended biological effect.

A therapeutically effective amount of an anti-tau antibody orepitope-binding fragment thereof of the invention may vary according tofactors such as the disease state, age, sex, and weight of theindividual, and the ability of the anti-tau antibody, or epitope-bindingfragment thereof, to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of the antibody or antibody portion are outweighedby the therapeutically beneficial effects.

As indicated above, the present invention particularly relates tomonoclonal antibodies, or epitope-binding fragments thereof, and to acompletely new method for producing such molecules (and thus of suchepitope-binding fragments thereof). This ability of the new method toisolate monoclonal antibodies is exemplified herein by its use toisolate monoclonal antibodies that are capable of specifically bindingto the phosphorylated residue serine 396 (P-S396, pS396, ^({p})S396) ofhuman tau (SEQ ID NO:1). These antibodies are further characterized bytheir ability to discriminate between phosphorylated residues serine 396and serine 404 (P-S404, pS404) such that they do not bind to tau proteinwith phosphorylated serine 404 unless the tau is also phosphorylated atresidue 396.

The antibodies of the present invention, or epitope-binding fragmentthereof, have been generated and isolated by use of a novel a methodwhich favors the selection of pS396 specific antibodies. Furthermore, byapplying this very strict antibody clone selection procedure, antibodieshave been obtained that are not only highly specific towards S396, butalso highly selective towards the phosphorylated pS396 epitope. Theseantibodies uniquely recognize tau from Alzheimer's disease brains. Thescreening procedure ensures the identification of antibodies whichpossess a functional and therapeutic utility.

Antibodies were raised against the bi-phosphorylated peptide:TDHGAEIVYK{p}SPVVSGDT{p}SPRHL (SEQ ID NO:2) covering residues 386-408 of2N4R tau. Mice were immunized with the phospho-peptide. Once sufficientantibody titres had been obtained, the mice were sacrificed andhybridomas were generated. The hybridomas were screened using dot-blotand MSD ELISA with immobilized human pathological and non-pathologicaltau. The ability to discriminate between pathological andnon-pathological human tau protein in dot-blot and Western blot was usedfor the selection of hybridomas. Sixteen clones were selected, of whichfour hybridoma clones were recovered that produced antibodies whichexhibited extraordinary capabilities for binding to human pathologicaltau material.

Specific binding to pathological and non-pathological tau was alsodetermined by isolation of tau from diseased and non-diseased human ADbrains and immobilization of this material on MSD ELISA plates (Example4).

A further aspect of the invention relates to monoclonal antibody or anepitope-binding fragment thereof elicited against the bi-phosphorylatedpeptide comprising at least 18, such as at least 20 amino consecutiveacid residues within TDHGAEIVYK^({p})SPVVSGDT^({p})SPRHL (SEQ ID NO:2)covering residues 386-410 of 2N4R tau. In this aspect of the invention,the monoclonal antibody or an epitope-binding fragment thereof istypically elicited against the bi-phosphorylated peptide comprising18-40, such as at 18-30, such as 20-30 amino consecutive acid residuescomprising TDHGAEIVYK^({p})SPVVSGDT^({p})SPRHL (SEQ ID NO:2) coveringresidues 386-410 of 2N4R tau.

A further aspect of the invention is directed to the monoclonal antibodyor an epitope-binding fragment thereof of the invention, having aspecificity for phosphoTau (pTau) from AD-diseased patients overage-matched healthy controls, such that said monoclonal antibody or anepitope-binding fragment thereof has a specificity difference forphosphoTau (pTau) from AD-diseased patients over tau from age-healthymatched controls of more than 50-fold, such as more than 100-foldincrease in specificity for AD disease material compared to healthycontrol material in an ELISA based assay detect phosphoTau (pTau) inbrain homogenates from AD and from healthy control subjects, using aphospho- and multimer-specific Setup 1 ELISA or MSD as described herein.

A related aspect of the invention is directed to the monoclonal antibodyor an epitope-binding fragment thereof of the invention, having aspecificity for AD-diseased Tau such that said monoclonal antibody or anepitope-binding fragment thereof has a specificity difference for ADover age-healthy matched controls of more than 50-fold, such as morethan 100-fold increase in specificity for AD disease material comparedto healthy control material in an ELISA or MSD based assay detectphosphoTau (pTau) in brain homogenates from AD and from healthy controlsubjects, using a phospho- and multimer-specific Setup 1 ELISA.

Setup 1 ELISA or MSD method comprises the steps A) a capture ofpathological human Tau antigens from AD brains using C10-2 coatedplates; B) incubation of Tau antigens with increasing concentrations ofpS396 specific antibodies; and C) detection of the residual free Tauantigen captured by immobilized C10.2 using sulfo-tagged anti human(total) Tau antibodies from MSD.

More specifically, step A comprises: coating of MSD plates (typicallyovernight at 4 degrees C.) with C10-2 antibody, typically 0.5 μg/ml(capture antibody) in coating buffer, blocking (typically 1 hour at roomtemperature) and washing, typically 3 times. Step B comprises: mixing ofsamples of P3 lysate (typically diluted 1:1000=2-4 μg/ml total protein)and/or S1(p) (typically diluted 1:300=20-40 ng/ml total protein) from AD(pooled from 3 patients) with graded concentrations of pS396 peptideepitope specific antibody and incubating (typically 1 hour at roomtemperature). The reactions are subsequently incubated for 2 hours onplates prepared in step A. Step C comprises detecting C10-2 captured Tauwas using sulfo-tagged human tau antibody (typically 1:50) from MSDfollowing manufacturer's instructions. Plates are analyzed on MSDSECTOR® S600. AD P3 and AD S1(p) are tested in a similar setup.

A further embodiment is directed to an antibody, or antigen-bindingfragment thereof, capable of specifically binding to the phosphorylatedserine residue 396 of human tau (SEQ ID NO:1), which has been producedor manufactured in a cell line such as a human cell line, a mammalnon-human cell line, an insect, yeast or bacterial cell line.

The antibody, or antigen binding fragment thereof, capable ofspecifically binding to the phosphorylated residue 396 of human tau (SEQID NO:1), produced in a CHO cell line, HEK cell line, BHK-21 cell line,murine cell line (such as a myeloma cell line), fibrosarcoma cell line,PER.C6 cell line, HKB-11 cell line, CAP cell line and HuH-7 human cellline.

Specific affinities and binding properties of C10-2 and C10.2 Variantshave been characterized using tau 386-410 (2R4N) peptides which areeither phosphorylated or un-phosphorylated at position 396 or 404. Usingthe specific immunization and screening protocol outlined in thisapplication will produce highly phosphor-serine-396 (pS396) specificantibodies.

In order to demonstrate that the antibodies are specific towardspathological tau, C10-2 antibodies have also been characterized byimmune-histochemical analysis. The antibodies exhibit highly specificbinding to neurofibrillary tangles in Alzheimer's disease brains (tautangles) and in sections from Tg4510 tau transgenic mice expressinghuman (P301L) mutant tau. No binding is observed to tissue from humancontrol brains and from non-transgenic mouse brains, demonstrating thatthe antibodies specifically bind human tau and in particular tauassociated with Alzheimer's pathology.

Antibody C10-2

One aspect of the invention is directed to a monoclonal antibody, orepitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

A further aspect of the invention is directed to a monoclonal antibody,or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:12;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:11.

Alternatively defined, using the IMGT definition, one aspect of theinvention is directed to a monoclonal antibody, or epitope bindingfragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:40;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:41;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:42;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:43;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:44;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:45.

Alternatively defined, using the Chotia definition, one aspect of theinvention is directed to a monoclonal antibody, or epitope bindingfragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:52;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:53;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:54.

The unique capability of the antibodies of the invention to recognizetau associated with disease pathology is demonstrated. The binding ofpathological vs. non-pathological tau was compared. The comparison is tofive published tau antibodies: hACl-2B6, IPN002, HJ8.5, 2.10.3, and 4E4.2.10.3 antibody is a commercially available recombinant monoclonalantibody that specifically binds to Tau that is phosphorylated at serine422 (pS422). HJ8.5 is a commercially available monoclonal antibody thatrecognizes only human tau at the N-terminal region (epitope at residues25-30 aa). The antibody is of the IgG2b isotype. Anti-Tau TherapeuticAntibody NI-105-4E4i s a commercially available antibody. The tabledemonstrates that the isolated antibodies exhibit an exceptionally highdegree of specificity and selectivity towards human pathological tau.This selectivity is superior to any of the comparator antibodies asshown in Table 5.

TABLE 5 mAb Tested AD/ctrl TG/wt hACI-2B6 3 1 IPN002 3 37 HJ8.5 3 51 4E4no binding 1 2.10.3 5 2 Antibody C10-2 >100   118

At saturation, Antibody C10-2 exhibits more than 100-fold selectivitytowards P3 tau isolated from human AD brains.

To demonstrate that the selected antibodies have functional andtherapeutic utility, antibodies were tested in in-vitro and in-celltau-aggregation assays (Example 8). These assays are functional assayswhich demonstrate that the antibodies are able to interfere with thepathological aggregation process of tau. HEK293 cells are transientlytransfected with human tau-P301L-FLAG (4R0N). Subsequently the cells areexposed to tau extracts from human AD brains or from transgenic Tg4510brains. This exposure to pathological tau promotes tau uptake into cellsand intracellular aggregation. Both immunodepletion of tau-preparationsusing Antibody C10-2, and direct treatment of cells with theseantibodies is able to reduce the formation of tau aggregatesdramatically.

Therapeutic utility of Antibody C10-2 has also been evaluated in thehuman tau/PS1 mouse. This mouse model is a more AD disease relevantanimal model which only generates AD pathology late in life (12-18 monthof age). However, the mice do exhibit tau hyper phosphorylation beforethe occurrence of solid tangle pathology. Mice were injected chronicallyfor 13 weeks, twice weekly with 15 mg/kg dose. Antibody treated miceexhibit a dramatic reduction in phosphorylated tau, indicating thatchronic treatment with C10-2 will reduce tangle pathology and thussubsequent neurodegeneration in vivo.

The antibodies of the invention specifically remove hyperphosphorylatedtau from rTg4510 mouse brain extracts by immunodepletion methods.Moreover, the antibodies of the invention do not remove the normal taufrom the homogenates, whereas the commercially available tau5 antibodydoes. In contrast to commercial antibodies which bind to tau proteinswherein phosphorylation at residue 404 or at both residues 404 and 396,the antibodies of the invention specifically remove thehyperphosphorylated tau by 95% that is phosphorylated on serine 396.Experiments (Example 12) demonstrate that the antibody of the invention,despite only removing a very small fraction of the total tau in thebrain homogenate (8%), the antibodies do however specifically remove thehyperphosphorylated tau (by 90%). Accordingly, one aspect of theinvention is directed to a monoclonal antibody, or an epitope-bindingfragment thereof, capable of specifically binding to the pathogenichyperphosphorylated tau. Furthermore, in experiments whereinhyperphosphorylated Tau was removed using an antibody of the invention,the seeding activity is abolished. By removing hyperphosphorylated taufrom the homogenates, the homogenates no longer induce seeding of Taupathology. It has been proposed that reduction of seeding reduces thedevelopment of tangle formation and the progression of tauopathies,including Alzheimer's disease. Accordingly, a further aspect of theinvention is directed to an antibody of the invention for use in thereduction of the progression of AD or in the symptoms of AD.

Antibody D55E

One aspect of the invention relates to the variant antibody of AntibodyC10-2, Antibody D55E. This aspect of the invention is directed to amonoclonal antibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:28and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

A further embodiment of the aspect of the invention directed to AntibodyD55E relates to a monoclonal antibody, or epitope binding fragmentthereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:12:(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:13.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody D55E relates to a monoclonal antibody, orepitope binding fragment thereof, comprising at least one of

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:28and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Antibody D55Q

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody D55Q. This aspect of the invention is directedto a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

A further embodiment of the aspect of the invention directed to AntibodyD55Q relates to a monoclonal antibody, or epitope binding fragmentthereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:12;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:14.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody D55Q relates to a monoclonal antibody, orepitope binding fragment thereof, comprising at least one of

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Antibody D55S

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody D55S. This aspect of the invention is directedto a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:30;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

A further embodiment of the aspect of the invention directed to AntibodyD55S relates to a monoclonal antibody, or epitope binding fragmentthereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:12 and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:15.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody D55S relates to a monoclonal antibody, orepitope binding fragment thereof, comprising at least one of

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:30;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.Studies using Antibody D55S, Antibody D55Q, Antibody D55E indicates thatmutation of this residue results in an antibody with unaltered bindingproperties when comparing said antibodies prior to and subsequent totreatment with low pH for an extended period of time at roomtemperature, indicating that no isomerization is taken place at low pHor that the any isomerized protein has unaltered binding propertiescompared to pre-treatment.

Antibody N32S

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N32S. This aspect of the invention is directedto a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:31;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

A further embodiment of the aspect of the invention directed to AntibodyN32S relates to a monoclonal antibody, or epitope binding fragmentthereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:16;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:11.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32S relates to a monoclonal antibody, orepitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:31;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

An alternative definition of Antibody N32S, using the IMGT definition,is a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:46;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:47;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:48;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:43;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:44;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:45.

A further alternative definition of Antibody N32S, using the Chotiadefinition, is a monoclonal antibody, or epitope binding fragmentthereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:31;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:52;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:53;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:54.

As can be seen from FIG. 1, the IC50 of Antibody N32S (black circles) isreduced compared to Antibody C10-2: the IC50 of N32S was calculated tobe 44 nM. This notable improvement over C10-2 was not anticipated. Assupported by FIG. 1, one aspect of the invention is directed to anantibody that inhibits AD-P3 in the fluid phase inhibition assaydescribed herein, such that the signal is reduced by 50% at aconcentration of 100 nM or less of the antibody. In one embodiment, theantibody of the invention has an IC50 of from 0.1 nM to 100 nM, such asat a concentration of 50 nM or less, such as from 0.1 nM to 50 nM, basedon fluid phase inhibition assay for AD-P3 capture.

The data generated using Antibody N32S indicates that mutation atposition 32 of the Light Chain of Antibody C10-2, or of the LC CDR1 ofAntibody C10-2 to serine results in increased apparent affinity (1050)in peptide inhibition assays. Moreover, mutation at position 32 toserine or glutamine (Antibody N32S and Antibody N32Q) abolishesdeamidation in both positions 32 and 34 as shown in FIG. 20. Potency, asdetermined in terms of in-vitro seeding and aggregation studies, ismaintained in both N32S and N32Q variants.

The potential deamidation potential identified in C 10-2 at positionwhich could lead to heterogeneity of protein batches. C 10-2 showedheterogenous binding to AD-P3 antigens with a minor fraction of highapparent affinity (2-5 nM) and a predominant binding with low apparentaffinity (200-1000 nM) (FIG. 1). The heterogenous binding could reflectdifferent subpopulations of deamidated and non-deamidated C 10-2. Avariant was generated by substitution (N32S) which preventeddeamidation. As shown improved binding by activity as indicated byoverall reduced IC50 (higher apparent affinity) compared to C 10-2. Anadditional substitution A101T was introduced resulting in homogenousbinding of high apparent affinity type. The improved binding activity inboth the N32S and N32S-A101T suggest more stable and homogenousantibodies was achieved by mutations described above.

Antibody N32Q

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N32Q. This aspect of the invention is directedto a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:32;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

A further embodiment of the aspect of the invention directed to AntibodyN32Q relates to a monoclonal antibody, or epitope binding fragmentthereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:17;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:11.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32Q relates to a monoclonal antibody, orepitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:32;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

As stated, the data generated using Antibody N32S indicates thatmutation at position 32 of the Light Chain of Antibody C10-2, or of theLC CDR1 of Antibody C10-2 to serine results in increased apparentaffinity (IC50), whereas mutation to glutamine (Antibody N32Q) resultsin unaltered binding activity. However, mutation at position 32 toserine or glutamine (Antibody N32S and Antibody N32Q) abolishesdeamidation in both positions 32 and 34 as shown in FIG. 20.Accordingly, there are advantages to both Antibody N32S and AntibodyN32Q. Potency, as determined in terms of in-vitro seeding andaggregation studies, is maintained in both N32S and N32Q variants.

Antibody N34S

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N34S. This aspect of the invention is directedto a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:33;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

A further embodiment of the aspect of the invention directed to AntibodyN34S relates to a monoclonal antibody, or epitope binding fragmentthereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:18;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:11.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N34S relates to a monoclonal antibody, orepitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:33;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Antibody N34Q

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N34Q. This aspect of the invention is directedto a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:34;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

A further embodiment of the aspect of the invention directed to AntibodyN34Q relates to a monoclonal antibody, or epitope binding fragmentthereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:19;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:11.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N34Q relates to a monoclonal antibody, orepitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:34;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Antibody N32S, N34S

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N32S, N34S. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:35;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32S, N34S relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:20;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:11.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32S, N34S relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:35;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Antibody N32Q, N34S

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N32Q, N34S. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:36;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32Q, N34S relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:21;(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:11.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32Q, N34S relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:36;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Antibody N32Q, N34Q

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N32Q, N34Q. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:37;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8;

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32Q, N34Q relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:22;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:11.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32Q, N34Q relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:37;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Antibody N32S, N34Q

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N32S, N34Q. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:38;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32S, N34Q relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:23;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:11.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32S, N34Q relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:38;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Antibody A101T

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody A101T. This aspect of the invention is directedto a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody A101T relates to a monoclonal antibody,or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:12;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:24.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody A101T relates to a monoclonal antibody,or epitope binding fragment thereof, comprising at least one of

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

The data generated using Antibody A101T indicates that the mutation ofHeavy Chain of Antibody C10-2 or of the Heavy Chain CR3 of AntibodyC10-2 results in a two-fold increased peptide binding and a 10-20 foldincreased binding to P3 material. Furthermore, variants comprising theA101T mutation have increased potency in in-vitro seeding assays.

Antibody N32S, A101T

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N32S, A101T. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:31;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32S, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:16;(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:24.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32S, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:31;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39;and further comprising at least one of(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;and(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7.

Using the IMGT definition, Antibody N32S, A101T is a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:46;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:47;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:48;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:49;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:50;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:51.

Using the Chotia definition, Antibody N32S, A101T is a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:31;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:55;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:56;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:57.

As can be seen from FIG. 1, the IC50 of Antibody N32S, A101T (whitecircles) is dramatically reduced compared to Antibody C10-2: the IC50 ofN32S A101T was calculated to be 14 nM. This notable improvement overC10-2 was not anticipated. Based upon FIG. 1, one aspect of theinvention is directed to an antibody that inhibits AD-P3 in the fluidphase inhibition assay described herein, such that the signal is reducedby 50% at a concentration of 100 nM or less of the antibody, such asfrom 10 nM to 100 nM of the antibody, such as at a concentration of 50nM or less, such as from 10 nM to 50 nM of the antibody. As shown inFIG. 20, Antibody N32S, A101 is very stable against deamidation. Asshown in FIG. 19, Antibody N32S, A101T showed a stronger reduction inaggregation.

Antibody N32Q, A101T

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N32Q, A101T. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:32;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32Q, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:17;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:24.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32Q, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:32;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39;and further comprising at least one of(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;and(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7.

Antibody N32S, D55E

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N32S, D55E. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:31;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:28;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32S, D55E relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:16;(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:13.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32S, D55E relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:31;and(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:28;and further comprising at least one of(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Antibody N32Q, D55E

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N32Q, D55E. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:32;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:28;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32Q, D55E relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:17;(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:13.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N32Q, D55E relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:32;and(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:28;and further comprising at least one of(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.

Antibody N34S, A101T

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N34S, A101T. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:33;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N34S, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:18;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:24.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N34S, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:33;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39;and further comprising at least one of(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;and(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7.

Antibody N34Q, A101T

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody N34Q, A101T. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:34;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N34Q, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:19;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:24.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody N34Q, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:34;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39;and further comprising at least one of(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising at least one of(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7.

Antibody D55E, A101T

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody D55E, A101T. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:28;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody D55E, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:12;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:25.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody D55E, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising at least oneof

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:28;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

Antibody D55Q, A101T

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody D55Q, A101T. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody D55Q, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:12;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:26.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody D55Q, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising at least oneof

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:29;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

Antibody D55S, A101T

Another aspect of the invention relates to the variant antibody ofAntibody C10-2, Antibody D55S, A101T. This aspect of the invention isdirected to a monoclonal antibody, or epitope binding fragment thereof,comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:30;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody D55S, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising

(a) a Light Chain comprising the amino acid sequence of SEQ ID NO:12;and(b) a Heavy Chain comprising the amino acid sequence of SEQ ID NO:27.

Alternatively defined, a further embodiment of the aspect of theinvention directed to Antibody D55S, A101T relates to a monoclonalantibody, or epitope binding fragment thereof, comprising at least oneof

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;and(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and further comprising(d) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(e) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:30;and(f) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.

Combinations of the Heavy Chains Variants and Light Chain Variants areanticipated, such as multiple variants within a Light Chain and/ormultiple variants within a Heavy Chain are anticipated, a combination ofa singular variant within a Light Chain with a singular or multiplevariant within a Heavy Chain, combinations of multiple variants within aLight Chain with multiple variants within a Heavy Chain. The antibody ofthe invention preferably comprises

a Light Chain selected from the group consisting of SEQ ID NO:12 (LightChain C10-2); SEQ ID NO:16 (Light Chain Variant N32S); SEQ ID NO:17(Light Chain Variant N32Q); SEQ ID NO:18 (Light Chain Variant N34S); SEQID NO:19 Light Chain Variant N34Q); SEQ ID NO:20 (Light Chain VariantN32S, N34S); SEQ ID NO:21 (Light Chain Variant N32Q, N34S); SEQ ID NO:22(Light Chain Variant N32Q, N34Q); and SEQ ID NO:23 (Light Chain VariantN32S, N34Q); anda Heavy Chain selected from the group consisting ofSEQ ID NO:11 (Heavy Chain C10-2); SEQ ID NO:13 (Heavy Chain VariantD55E); SEQ ID NO:14 (Heavy Chain Variant D55Q); SEQ ID NO:15 (HeavyChain Variant D55S); SEQ ID NO:24 (Heavy Chain Variant A101T); SEQ IDNO:25 (Heavy Chain Variant D55E, A101T); SEQ ID NO:26 (Heavy ChainVariant D55Q, A101T), and SEQ ID NO:27 (Heavy Chain Variant D55S,A101T).

In one embodiment, when the Light Chain is SEQ ID NO:12, the Heavy Chainis selected from the group consisting of SEQ ID NO:13 (Heavy ChainVariant D55E); SEQ ID NO:14 (Heavy Chain Variant D55Q); SEQ ID NO:15(Heavy Chain Variant D55S); SEQ ID NO:24 (Heavy Chain Variant A101T);SEQ ID NO:25 (Heavy Chain Variant D55E, A101T); SEQ ID NO:26 (HeavyChain Variant D55Q, A101T), and SEQ ID NO:27 (Heavy Chain Variant D55S,A101T). In an alternative embodiment, when the Heavy Chain is SEQ IDNO:11, the Light Chain is selected from the group consisting of SEQ IDNO:16 (Light Chain Variant N32S); SEQ ID NO:17 (Light Chain VariantN32Q); SEQ ID NO:18 (Light Chain Variant N34S); SEQ ID NO:19 Light ChainVariant N34Q); SEQ ID NO:20 (Light Chain Variant N32S, N34S); SEQ IDNO:21 (Light Chain Variant N32Q, N34S); SEQ ID NO:22 (Light ChainVariant N32Q, N34Q); and SEQ ID NO:23 (Light Chain Variant N32S, N34Q).

The monoclonal antibody, or epitope-binding fragment thereof, suitablycomprises

(a) a Light Chain CDR1 comprising the amino acid sequence selected fromthe group consisting of SEQ ID NO:3, SEQ ID NO:31; SEQ ID NO:32; SEQ IDNO:33; SEQ ID NO:34; SEQ ID NO:35; SEQ ID NO:36; SEQ ID NO:37; and SEQID NO:38; (b) a Light Chain CDR2 comprising the amino acid sequence ofSEQ ID NO:4; (c) a Light Chain CDR3 comprising the amino acid sequenceof SEQ ID NO:5; (d) a Heavy Chain CDR1 comprising the amino acidsequence of SEQ ID NO:6; (e) a Heavy Chain CDR2 comprising the aminoacid sequence selected from the group consisting of SEQ ID NO:7; SEQ IDNO:28; SEQ ID NO:29; and SEQ ID NO:30; and (f) a Heavy Chain CDR3comprising the amino acid sequence selected from the group consisting ofSEQ ID NO:8, and SEQ ID NO:39.

Interesting embodiments of combinations of variants include wherein theantibodies are selected from the group consisting of Antibody N32S,A101T; Antibody N32Q, A101T; Antibody N32S, D55E; and Antibody N32Q,D55E, preferably Antibody N32S, A101T. As can be seen from the Examples,Antibody N32S and Antibody N32S, A101T are preferred embodiment.

Altogether, the Examples show that the antibodies of the invention,including C10-2, bind efficiently to AD-P3 antigens coated MSD plates.In comparison, commercial antibodies such as PHF-13, have low bindingactivity. Furthermore PHF-13 demonstrated substantial higher degree ofnon-specific binding in comparison to the antibodies of the invention.C10-2 fluid phase inhibition of Ptau antigen capture in C10-2 coatedplate is effective (1050=10-20 nM) whereas PHF-13 is ineffective(IC50=500-1000 nM).

One aspect of the invention is directed to an antibody comprising

(a) a Light Chain CDR1 having the amino acid sequence of SEQ ID NO:3;(b) a Light Chain CDR2 having the amino acid sequence of SEQ ID NO:4;(c) a Light Chain CDR3 having the amino acid sequence of SEQ ID NO:5;and(d) a Heavy Chain selected from the group consisting of SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24, SEQ ID NO:25,SEQ ID NO:26, and SEQ ID NO:27.

One aspect of the invention is directed to an antibody comprising

(a) a Heavy Chain CDR1 having the amino acid sequence of SEQ ID NO:6;(b) a Heavy Chain CDR2 having the amino acid sequence of SEQ ID NO:7;(c) a Heavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8;and(d) a Light Chain selected from the group consisting of SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, and SEQ ID NO:23.

The monoclonal antibody, or epitope-binding fragment thereof, of theinvention, typically inhibits AD-P3 in the fluid phase inhibition assaysuch that the signal is reduced by 50% at a concentration of 100 nM orless of the antibody, such as from 10 nM to 100 nM of the antibody, suchas at a concentration of 50 nM or less, such as from 10 nM to 50 nM ofthe antibody. The monoclonal antibody, or epitope-binding fragmentthereof, of the invention, typically, according to Western Blot signalof pS396 tau after immunodepletiion studies on Alzheimers diseased brainextracts, is capable of removing at least 15% tau phosphorylated atserine 396 from AD brain homogenates at about 75 ng of antibody.

The antibody or epitope-binding fragment thereof is preferably a humanor humanized antibody.

The antibodies and epitope-binding fragment thereof mentioned above may,according to one embodiment, further comprise a variant of such lightand/or heavy chain CDR1, CDR2 or CDR3 (with no more than 4 amino aciddifferences, or no more than 3 amino acid differences, or no more than 2amino acid differences, or no more than 1 amino acid difference.

As can be seen from FIG. 18, HC CDR1, HC CDR2, HC CDR3 and LC CDR3 are,in at least one embodiment, important, for the binding to the 392-398region of Tau. In one embodiment of the invention, the antibody of theinvention, or epitope-binding fragment thereof comprises: In one aspectof the invention, the invention is directed to an antibody orepitope-binding fragments thereof, that forms a hydrophobic pocketformed by L3:H3, L3:F8*, H1:H13, H2:Y1, H2:Y3 with Y394 of the taupeptide. In an embodiment, the invention is directed to an antibody thatcompetes with an antibody further described herein for forming ahydrogen bonding network between solvated ^({p})S396 and L3:T4, H1:R10,H1:T11, H3:R1, H3:T3; (*) L3:F8 is the C-terminal flanking frameworkresidue of CDR L3 (see FIG. 11).

As can be seen from the x-ray crystal structure, the antibody of theinvention binds with two levels of selectivity. The first level ofselectivity is selectivity for hyperphosphorylated pathological, tau andthe second level of selectivity is selectivity for a phosphorylatedserine residue wherein the phosphate of said phosphorylated serine ishydrogen bonded to the side chain of a tyrosine residue separated by oneresidues from said phosphorylated serine. Accordingly, an interestingaspect of the invention is directed to a monoclonal antibody, orepitope-binding fragment thereof, selective for an amino acid motif ofhyperphosphorylated tau whose motif comprises of a phosphorylated serineresidue and tyrosine residue spaced by a single residue. Typically, theamino acid motif has the sequence:

Y-X-S(phosphorylated)-P-

wherein Y is tyrosine, X is a naturally occurring amino acid, P isproline andS(phosphorylated) is serine with a phosphorylated hydroxyl side chain.

Similarly, an interesting aspect of the invention is directed to anantibody or epitope-binding fragment thereof which binds tophosphorylated tau, preferably hyperphosphorylated tau, wherein saidantibody or epitope-binding fragment thereof is selective for the aminoacid residue motif IA, wherein R is a side chain of a naturallyoccurring amino acid.

Without being bound to a particular theory, it is believed that theantibody of the invention is selective for amino acid motif IA when saidmotif is in a conformation adopted by pathological tau. Accordingly, theamino acid motif IA is typically the sequence selectively recognized bythe antibody of the invention. Accordingly, an interesting aspect of theinvention is directed to an antibody or epitope-binding fragment thereofwhich binds to phosphorylated tau, preferably hyperphosphorylated tau,wherein said antibody or epitope-binding fragment thereof is selectivefor the amino acid residue motif IA, wherein R is a side chain of anaturally occurring amino acid.

In a typical embodiment of this aspect of the invention, the inventionis directed to an antibody or epitope-binding fragment thereof whichbinds to phosphorylated tau, preferably hyperphosphorylated tau, whereinsaid antibody or epitope-binding fragment thereof is selective for theamino acid residue motif IB, wherein R is a side chain of a naturallyoccurring amino acid such as, but not limited to IC or ID.

One aspect of the invention, the HC CDR1 region of the antibody of theinvention, Asp Arg Thr Ile His (SEQ. ID. NO. 7) interacts with themotifs IA-ID of hyperphosphorylated tau.

As can be seen from FIG. 18, in this aspect of the invention, thepresence of two consecutive charged residues, namely aspartic acid andarginine, in the HC CDR1 play a role in hydrogen bonding with the motifwithin the targeted tau epitope wherein the antibody is selective for anamino acid motif of hyperphosphorylated tau whose motif comprises of aphosphorylated serine residue and tyrosine residue spaced by a singleresidue. Accordingly, one aspect of the invention is directed to anantibody or epitope-binding fragment thereof which binds tophosphorylated tau, preferably hyperphosphorylated tau, wherein saidantibody or epitope-binding fragment thereof is selective for the aminoacid residue motif IA-ID wherein the antibody comprises a HC CDR1 regioncomprises two consecutive charged amino acid residues, such as asparticacid and arginine. In a further alternative of this aspect of theinvention, the antibody or epitope-binding fragment thereof of inventionbinds to hyperphosphorylated tau, wherein said antibody orepitope-binding fragment thereof is selective an epitope comprisingSerine 396 and Tyrosine 394, wherein a PO4 group forms a covalent bondwith one of said Serine 396 and Tyrosine 394 and a hydrogen bond withthe other of Serine 396 and Tyrosine 394 wherein the antibody comprisesa HC CDR1 regions comprising two consecutive charged residues, such asaspartic acid and arginine. The charged amino acid residues of the HCCDR1 region may be selected from the group consisting of arginine,lysine, aspartic acid and glutamic acid wherein at least one of saidresidues is aspartic acid or arginine, preferably wherein one chargedresidue is arginine and the other is aspartic acid.

In a further embodiment of this aspect of the invention, one or both ofthe two consecutive charged amino acid residues are flanked by a polaramino acid residue, preferably selected from threonine and tyrosine.Furthermore, one or both of the one or both of the two consecutivecharged amino acid residues are flanked by a 3-amino acid residue motifof polar residue-hydrophobic residue-polar residue.

In one embodiment, HC CDR1 comprises the 5-residue motif POLARAA-HYRDROPHOBIC AA-POLAR AA-CHARGED AA-CHARGED AA, wherein at least oneof said residues is aspartic acid or arginine, preferably wherein onecharged residue is arginine and the other is aspartic acid. Preferably,the 5-residue motif has the motif POLAR AA-HYRDROPHOBIC AA-POLARAA-Asp-Arg. More preferably, the 5-residue motif of the HC CDR1 has thesequence TFTDR (SEQ ID NO:58).

It is interesting to note that the HC CDR1 of the antibody of theinvention comprises the motif POLAR-HYRDROPHOBIC-POLAR-CHARGED on bothsides of the two consecutive charged amino acid residues involved inhydrogen bonding with the phosphate group involved in the electrostaticinteraction between S396 and Y394. Accordingly, in the preferredembodiment, the HC CDR1 of invention comprises the palindromic 8-residuemotif POLAR AA-HYRDROPHOBIC AA-POLAR AA-CHARGED AA-CHARGED AA-POLARAA-HYRDROPHOBIC AA-POLAR AA. Preferably, the 8-residue motif of HC CDR1comprises the motif TFTDR (SEQ ID NO:58)-POLAR AA-HYRDROPHOBIC AA-POLARAA. More preferably, the 8-residue motif of HC CDR1 comprises the motifTFTDRTIH (SEQ ID NO:59).

In one embodiment, the antibody recognizes an epitope within residue392-398 of hyperphopshorylated tau comprising Serine 396 and Tyrosine394 wherein Serine 396 is phosphorylated and wherein the antibodycomprises a HC CDR1 region comprising two consecutive charged amino acidresidues. In a preferred embodiment, the antibody recognizes an epitopewithin residue 392-398 of hyperphopshorylated tau comprising Serine 396and Tyrosine 394 wherein Serine 396 is phosphorylated and wherein theantibody comprise a HC CDR1 region comprises the 5-residue motif POLARAA-HYRDROPHOBIC AA-POLAR AA-CHARGED AA-CHARGED AA. In a more preferredembodiment, the antibody recognizes an epitope within residue 392-398 ofhyperphopshorylated tau comprising Serine 396 and Tyrosine 394 whereinSerine 396 is phosphorylated and wherein the antibody comprises a HCCDR1 region comprising the motif POLAR AA-HYRDROPHOBIC AA-POLARAA-CHARGED AA-CHARGED AA-POLAR AA-HYRDROPHOBIC AA-POLAR AA.

In a further preferred embodiment, the antibody comprises a HC CDR1region as defined herein and a HC CDR3 region comprising a 6-residuemotif comprising at least two charged residues.

The present invention also provides a method of reducing tau tangleformation in a patient, comprising administering to the patient in needof such treatment, a therapeutically effective amount of an antibody ofthe invention, or epitope-binding fragments thereof.

One aspect of the invention is directed to a method of treating ataupathy using an antibody of the invention, or epitope-bindingfragments thereof. Typically, the taupathy is selected from the groupconsisting of Alzheimer's disease, Argyrophilic Grain Disease (AGD),Psychosis, particularly Psychosis due to AD or Psychosis in patientswith AD, psychiatric symptoms of patients with Lewy body dementia,Progressive Supranuclear Palsy (PSP), Frontotemporal dementia (FTD orvariants thereof), TBI (traumatic brain injury, acute or chronic),Corticobasal Degeneration (CBD), Picks Disease, Primary age-relatedtauopathy (PART), Neurofibrillary tangle-predominant senile dementia,Dementia pugilistica, Chronic traumatic encephalopathy, stroke, strokerecovery, neurodegeneration in relation to Parkinson's disease,Parkinsonism linked to chromosome, Lytico-Bodig disease(Parkinson-dementia complex of Guam), Ganglioglioma and gangliocytoma,Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosingpanencephalitis, Huntington's disease, lead encephalopathy, tuberoussclerosis, Hallervorden-Spatz disease and lipofuscinosis. Moretypically, the taupathy is selected from the group consisting ofAlzheimer's disease, Argyrophilic Grain Disease (AGD), Psychosis,particularly Psychosis due to AD or Psychosis in patients with AD,psychiatric symptoms of patients with Lewy body dementia, ProgressiveSupranuclear Palsy (PSP), Frontotemporal dementia (FTD or variantsthereof), TBI (traumatic brain injury, acute or chronic), CorticobasalDegeneration (CBD), and Picks Disease. In particular, the tauopathiesmay be selected from Alzheimer's disease, Argyrophilic Grain Disease(AGD), Psychosis due to AD or Psychosis in patients with AD, andpsychiatric symptoms of patients with Lewy body dementia.

Accordingly, a further aspect of the invention is directed to anantibody of the invention or epitope-binding fragments thereof, for usein the treatment of a taupathy. Typically, the taupathy is selected fromthe group consisting of Alzheimer's disease, Argyrophilic Grain Disease(AGD), Psychosis, particularly Psychosis due to AD or Psychosis inpatients with AD, psychiatric symptoms of patients with Lewy bodydementia, Progressive Supranuclear Palsy (PSP), Frontotemporal dementia(FTD or variants thereof), TBI (traumatic brain injury, acute orchronic), Corticobasal Degeneration (CBD), Picks Disease, Primaryage-related tauopathy (PART), Neurofibrillary tangle-predominant seniledementia, Dementia pugilistica, Chronic traumatic encephalopathy,stroke, stroke recovery, neurodegeneration in relation to Parkinson'sdisease, Parkinsonism linked to chromosome, Lytico-Bodig disease(Parkinson-dementia complex of Guam), Ganglioglioma and gangliocytoma,Meningioangiomatosis, Postencephalitic parkinsonism, Subacute sclerosingpanencephalitis, Huntington's disease, lead encephalopathy, tuberoussclerosis, Hallervorden-Spatz disease and lipofuscinosis. Moretypically, the taupathy is selected from the group consisting ofAlzheimer's disease, Argyrophilic Grain Disease (AGD), Psychosis,particularly Psychosis due to AD or Psychosis in patients with AD,apathy due to AD or apathy in patients with AD, psychiatric symptoms ofpatients with Lewy body dementia, Progressive Supranuclear Palsy (PSP),Frontotemporal dementia (FTD or variants thereof), TBI (traumatic braininjury, acute or chronic), Corticobasal Degeneration (CBD), and PicksDisease. In particular, the tauopathies may be selected from Alzheimer'sdisease, Argyrophilic Grain Disease (AGD), Psychosis due to AD orPsychosis in patients with AD, apathy due to AD or apathy in patientswith AD, and psychiatric symptoms of patients with Lewy body dementia.

One aspect of the invention is directed to a therapy comprising theadministration of i) a Tau antibody and ii) a compound selected from thegroup consisting of

a) a BACE inhibitor;b) a compound useful in active or passive Tau immunotherapy;c) a compound useful in active or passive AR peptide immunotherapy;d) an NMDA receptor antagonists;e) a further Tau protein aggregation inhibitor;e) an acetylcholine esterase inhibitor;f) an antiepileptic;g) an anti-inflammatory drug; and

h) an SSRI.

A further aspect of the invention is directed to a compositioncomprising i) a Tau antibody and ii) a compound selected from the groupconsisting of

a) a BACE inhibitor;b) a compound useful in active or passive Tau immunotherapy;c) a compound useful in active or passive AR peptide immunotherapy;d) an NMDA receptor antagonists;e) a further Tau protein aggregation inhibitor;e) an acetylcholine esterase inhibitor;f) an antiepileptic;g) an anti-inflammatory drug; and

h) an SSRI.

A further aspect of the invention is directed to a kit comprising i) acomposition comprising a Tau antibody and ii) a composition a compoundselected from the group consisting of

a) a BACE inhibitor;b) a compound useful in active or passive Tau immunotherapy;c) a compound useful in active or passive AR peptide immunotherapy;d) an NMDA receptor antagonists;e) a further Tau protein aggregation inhibitor;f) an acetylcholine esterase inhibitor;g) an antiepileptic;h) an anti-inflammatory drug; andi) an antidepressant.a) Tau Antibody Combined with a BACE 1 Inhibitor

In the therapy, composition or kit of the invention, a tau antibody maybe combined with a BACE 1 inhibitor. The BACE1 inhibitor may be a smallmolecule BACE I inhibitor such as LY2886721, MK-8931, AZD3293, or E2609.

In a further embodiment, the BACE 1 inhibitor is of the formula I

wherein Ar is selected from the group consisting of phenyl, pyridyl,pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl,isoxazolyl, and wherein Ar is optionally substituted with one or moresubstituents selected from halogen, CN, C1-C6 alkyl, C2-C6 alkenyl,C2-C6 alkynyl, C1-C6 fluoroalkyl or C1-C6 alkoxy; and R1 is one or morehydrogen, halogen, C1-C3 fluoroalkyl or C1-C3 alkyl; and R2 presentshydrogen or fluoro.

Exemplary compounds of Formula I include

Furthermore, a suitable BACE 1 inhibitor may be of Formula II

wherein Ar is selected from the group consisting of phenyl, pyridyl,pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, 1,2,4-triazolyl,thiophenyl, thiazolyl, oxazolyl, isoxazolyl, 1,3,4-thiadiazolyl,isothiazolyl, 1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, furazanyl and1,2,4-thiadiazolyl and where the Ar is optionally substituted with oneor more halogen, CN, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆fluoroalkyl or C₁-C₆ alkoxy; R¹ is C₁-C₃ alkyl or C₁-C₃ fluoroalkyl; R²is hydrogen, halogen, C₁-C₃ fluoroalkyl or C₁-C₃ alkyl; and R³ is C₁-C₃alkyl.

Examplary compounds of the BACE 1 inhibitor of Formula II include thecompounds selected from the group consisting of:N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-fluoropicolinamide;N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-methoxypyrazine-2-carboxamide;N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypicolinamide;N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-cyano-3-methylpicolinamide;andN-(3-((2R,5R)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide.

Other BACE inhibitors may be selected from the below formula

wherein Ar is selected from the group consisting of phenyl, pyridyl,pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl,isoxazolyl, and wherein Ar is optionally substituted with one or moresubstituents selected from halogen, CN, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₆ fluoroalkyl or C₁-C₆ alkoxy; andR¹ is one or more hydrogen, halogen, C₁-C₃ fluoroalkyl or C₁-C₃ alkyl;R² presents hydrogen or fluoro;or a pharmaceutically acceptable salt thereof.

For example, compounds such as

-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypicolinamide;-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)picolinamide;-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyano-3-methylpicolinamide;-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-chloropicolinamide;-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide;-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-2-methyloxazole-4-carboxamide;-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-4-bromo-1-methyl-1H-imidazole-2-carboxamide;-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-4-methylthiazole-2-carboxamide;-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-2-(difluoromethyl)oxazole-4-carboxamide;-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-1-(difluoromethyl)-1H-pyrazole-3-carboxamide;-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide;-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-4-chlorobenzamide;-   N-(3-((2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-fluoropicolinamide;-   N-(3-((2S,5R)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypicolinamide;-   N-[3-[(2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-3,4-dihydropyridin-2-yl]-4,    5-difluoro-phenyl]-5-fluoro-pyridine-2-carboxamide;-   N-[3-[(2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-3,4-dihydropyridin-2-yl]-4,    5-difluoro-phenyl]-5-methoxy-pyridine-2-carboxamide;-   N-[3-[(2S,5S)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-3,4-dihydropyridin-2-yl]-4,    5-difluoro-phenyl]-5-methoxy-pyrazine-2-carboxamide;-   N-[3-[(2S,5R)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-3,4-dihydropyridin-2-yl]-4,5-difluoro-phenyl]-5-fluoro-pyridine-2-carboxamide;-   N-[3-[(2S,5R)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-3,4-dihydropyridin-2-yl]-4,5-difluoro-phenyl]-5-methoxy-pyridine-2-carboxamide;-   N-[3-[(2S,5R)-6-amino-5-fluoro-5-(fluoromethyl)-2-methyl-3,4-dihydropyridin-2-yl]-4,5-difluoro-phenyl]-5-methoxy-pyrazine-2-carboxamide;-   N-[3-[(2S,5S)-6-amino-5-fluoro-2,5-bis(fluoromethyl)-3,4-dihydropyridin-2-yl]-4-fluoro-phenyl]-5-fluoro-pyridine-2-carboxamide;-   N-[3-[(2S,5S)-6-amino-5-fluoro-2,5-bis(fluoromethyl)-3,4-dihydropyridin-2-yl]-4-fluoro-phenyl]-5-methoxy-pyridine-2-carboxamide;-   N-[3-[(2S,5S)-6-amino-5-fluoro-2,5-bis(fluoromethyl)-3,4-dihydropyridin-2-yl]-4-fluoro-phenyl]-5-methoxy-pyrazine-2-carboxamide;-   N-[3-[(2S,5R)-6-amino-5-fluoro-2,    5-bis(fluoromethyl)-3,4-dihydropyridin-2-yl]-4-fluoro-phenyl]-5-fluoro-pyridine-2-carboxamide;-   N-[3-[(2S,5R)-6-amino-5-fluoro-2,    5-bis(fluoromethyl)-3,4-dihydropyridin-2-yl]-4-fluoro-phenyl]-5-methoxy-pyridine-2-carboxamide    and-   N-[3-[(2S,5R)-6-amino-5-fluoro-2,    5-bis(fluoromethyl)-3,4-dihydropyridin-2-yl]-4-fluoro-phenyl]-5-methoxy-pyrazine-2-carboxamide;    or a pharmaceutically acceptable salt of said compounds.

Other BACE inhibitors may be of the below formula

wherein Ar is selected from the group consisting of phenyl, pyridyl,pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl andisoxazolyl, and where the Ar is optionally substituted with one or morehalogen, CN, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6fluoroalkyl or C1-C6 alkoxy; andR1 and R2 independently are hydrogen, halogen, C1-C3 fluoroalkyl orC1-C3 alkyl; or a pharmaceutically acceptable salt thereof.

For example, compounds such as

-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-chloropicolinamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-fluoropicolinamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-1-(difluoromethyl)-1H-pyrazole-3-carboxamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-2-(difluoromethyl)oxazole-4-carboxamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyanopicolinamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-2-methyloxazole-4-carboxamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrimidine-2-carboxamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4,5-difluorophenyl)-2-methyloxazole-4-carboxamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4,5-difluorophenyl)-5-methoxypyrazine-2-carboxamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4,5-difluorophenyl)-5-fluoropicolinamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4,5-difluorophenyl)-5-chloropicolinamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4,5-difluorophenyl)-5-cyanopicolinamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4,5-difluorophenyl)-5-methoxypicolinamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4,5-difluorophenyl)-5-(methoxy-d3)picolinamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4,5-difluorophenyl)-5-cyano-3-methylpicolinamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)picolinamide(S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-bromopicolinamide-   (S)—N-(3-(6-amino-3,3-difluoro-2-(fluoromethyl)-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-bromopicolinamide    or a pharmaceutically acceptable salt of said compounds.

Other BACE compounds may be from the below formula

wherein Ar is selected from the group consisting of phenyl, pyridyl,pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl,isoxazolyl, and where the Ar is optionally substituted with one or moresubstituent selected from halogen, CN, C1-C6 alkyl, C2-C6 alkenyl, C2-C6alkynyl, C1-C6 fluoroalkyl or C1-C6 alkoxy; and

R1 is hydrogen, halogen, C1-C3 fluoroalkyl or C1-C3 alkyl;or a pharmaceutically acceptable salt thereof.

The compounds may be

-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-chloropicolinamide,-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-fluoropicolinamide,-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrazine-2-carboxamide,-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-2-methyloxazole-4-carboxamide,-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypicolinamide,-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide,-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyanopicolinamide,-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-4-methylthiazole-2-carboxamide,-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypyrimidine-2-carboxamide,-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxy-3-methylpyrazine-2-carboxamide,-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyano-3-methylpicolinamide,-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-bromopicolinamide,-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)picolinamide    and-   (S)—N-(3-(6-amino-2-(difluoromethyl)-3,3-difluoro-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)pyrazine-2-carboxamide;    or a pharmaceutically acceptable salt of said compounds.

Other BACE inhibitors may be of the below formula

wherein Ar is selected from the group consisting of phenyl, pyridyl,pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl, thiazolyl, oxazolyl,isoxazolyl, and where the Ar is optionally substituted with one or moresubstituent selected from halogen, CN, C1-C6 alkyl, C2-C6 alkenyl, C2-C6alkynyl, C1-C6 fluoroalkyl or C1-C6 alkoxy; andR1 is one or more hydrogen, halogen, C1-C3 fluoroalkyl or C1-C3 alkyl;or a pharmaceutically acceptable salt thereof.

The compound may be

-   N-(3-((2R,3S,5S)-6-amino-3,5-difluoro-2,5-dimethyl-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluoro-phenyl)-5-fluoropicolinamide,-   N-(3-((2R,3S,5S)-6-amino-3,5-difluoro-2,5-dimethyl-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluoro-phenyl)-5-methoxypyrazine-2-carboxamide,-   N-(3-((2R,3S,5S)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluoro-phenyl)-5-methoxypicolinamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluoro-phenyl)-5-fluoropicolinamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluoro-phenyl)-5-methoxypyrazine-2-carboxamide,-   N-(3-((2R,3S,5R)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluoro-phenyl)-2-methyloxazole-4-carboxamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluoro-phenyl)-5-methoxypicolinamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)picolinamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-chloropicolinamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-1-methyl-1H-imidazole-2-carboxamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(trifluoromethyl)pyrazine-2-carboxamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-4-methylthiazole-2-carboxamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-2-(difluoromethyl)oxazole-4-carboxamide,-   N-(3-((2R,3S,5R)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-1-(difluoromethyl)-1H-pyrazole-3-carboxamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-4-chlorobenzamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyanopicolinamide,-   N-(3-((2R,3S,5R)-6-amino-3, 5-difluoro-2,    5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4,5-difluorophenyl)-5-(methoxy-d3)picolinamide,-   N-(3-((2R,3S,5S)-6-amino-3,5-difluoro-2,5-dimethyl-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)picolinamide,-   N-(3-((2R,3S,5S)-6-amino-3,5-difluoro-2,5-dimethyl-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)pyrazine-2-carboxamide,-   N-(3-((2R,3S,5S)-6-amino-3,5-difluoro-2,5-dimethyl-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-cyano-3-methylpicolinamide,-   N-(3-((2R,3S,5S)-6-amino-3,5-difluoro-2,5-dimethyl-2,    3,4,5-tetrahydropyridin-2-yl)-4,5-difluorophenyl)-5-(methoxy-d3)picolinamide,-   N-(3-((2R,3R,5S)-6-amino-3,5-difluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-(methoxy-d3)picolinamide,-   N-(3-((2R,3S,5S)-6-amino-3,5-difluoro-2,5-dimethyl-2,    3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-bromopicolinamide    or a pharmaceutically acceptable salt of said compounds.    b) Tau Antibody Combined with a N3PGLU ABETA Antibody

In the therapy, composition or kit of the invention, a tau antibody maybe combined with a N3PGLU ABETA antibody.

c) Tau Antibody Combined with a Compound Useful in Active or Passive AβPeptide Immunotherapy

In the therapy, composition or kit of the invention, a tau antibody maybe combined with a compound useful in active or passive Aβ peptideimmunotherapy

d) Tau Antibody Combined with an NMDA Receptor Antagonist

In the therapy, composition or kit of the invention, a tau antibody maybe combined with an NMDA receptor antagonists. The NMDA receptorantagonist may be selected from the group consisting of, Memantine,Namenda, Namzaric (memantine/donepezil), and generic forms thereof. TheNMDA receptor antagonist may be selected from an antipsychotic.Presynaptic neurons prohibit excessive glutamate release via negativefeedback mechanisms, but such mechanisms are compromised underconditions of cellular stress, such as AD. Excess glutamate in thesynaptic cleft causes postsynaptic calcium channels to be continuallyopened, leading to increased intracellular calcium levels withinneurons, causing severe neuronal damage and/or death. By antagonizingthe NMDA receptor under conditions of excessive calcium inflow,antipsychotic reduce the excessive influx of calcium into neurons,decreasing cellular damage and improving normal neuronal signaling andthus cognitive function.

e) Tau Antibody Combined with a Further Tau Protein AggregationInhibitor;

In the therapy, composition or kit of the invention, a tau antibody maybe combined with a Tau protein aggregation inhibitor.

f) Tau Antibody Combined with an Acetylcholine Esterase Inhibitor

In the therapy, composition or kit of the invention, a tau antibody maybe combined with an acetylcholine esterase inhibitor (AChEI). AChEIs areoften used as a first-line treatment for cognitive symptoms in mild tomoderate AD. AChEIs are also used broadly to treat moderate to severeAD, including donepezil which is approved for this subpopulation. AChEIsalleviate the cholinergic deficit observed in AD patients, and improve apatient's ability to perform daily activities. In one embodiment, theinvention comprises a therapy comprising a tau antibody as defineherein, and an acetylcholine esterase inhibitor

In one embodiment, the AChEI is selected from the group consisting ofDonepezil, Galantamine and Rivastigmine. The AChEI may be an oraltablet, a jelly, a syrup, or othe form of oral solution formulation. TheAChEI may be also be a patch for transdermal administration.

g) Tau Antibody Combined with an Antiepileptic;

In the therapy, composition or kit of the invention, a tau antibody maybe combined with an antiepileptic.

h) Tau Antibody Combined with an Anti-Inflammatory Drug;

In the therapy, composition or kit of the invention, a tau antibody maybe combined with an anti-inflammatory drug.

i) Tau Antibody Combined with an an Antidepressant

In the therapy, composition or kit of the invention, a tau antibody maybe combined with an an antidepressant.

Depression is a common early comorbid symptom of AD dementia. Tricyclicantidepressants were once the preferred treatment for depressivesymptoms in AD, but SSRIs have largely replaced these agents. In oneembodiment, Escitalopram is the antidepressant since it is frequentlyprescribed for AD because of its favorable sideeffect profile andminimal drug interactions. In a further embodiment, citalopram orsertraline is the antidepressant as they are also frequently used. In afurther embodiment, vortioxetine is the antidepressant since it isrelated to improvement in cognitive performance and imaging evidence ofneural efficacy in patients suffering from MDD. The antidepressant maybe selected from the group consisting of Escitalopram, Sertraline,Citalopram, Paroxetine, Fluoxetine, Venlafaxine, Trazodone, Mirtazapine,Vortioxetine and generic forms thereof.

A further aspect of the invention is directed to an antibody of theinvention or epitope-binding fragments thereof, in a compositiontogether with a pharmaceutically acceptable carrier, diluent, adjuvantand/or stabilizer. The antibodies of the invention, or epitope-bindingfragments thereof, may be used in therapy for the treatment of ataupathy. Typically, the taupathy is selected from the group consistingof Alzheimer's disease, Argyrophilic Grain Disease (AGD), Psychosis,particularly Psychosis due to AD or Psychosis in patients with AD,apathy due to AD or apathy in patients with AD, psychiatric symptoms ofpatients with Lewy body dementia, Progressive Supranuclear Palsy (PSP),Frontotemporal dementia (FTD or variants thereof), TBI (traumatic braininjury, acute or chronic), Corticobasal Degeneration (CBD), PicksDisease, Primary age-related tauopathy (PART), Neurofibrillarytangle-predominant senile dementia, Dementia pugilistica, Chronictraumatic encephalopathy, stroke, stroke recovery, neurodegeneration inrelation to Parkinson's disease, Parkinsonism linked to chromosome,Lytico-Bodig disease (Parkinson-dementia complex of Guam), Gangliogliomaand gangliocytoma, Meningioangiomatosis, Postencephalitic parkinsonism,Subacute sclerosing panencephalitis, Huntington's disease, leadencephalopathy, tuberous sclerosis, Hallervorden-Spatz disease andlipofuscinosis. More typically, the taupathy is selected from the groupconsisting of Alzheimer's disease, Argyrophilic Grain Disease (AGD),Psychosis, particularly Psychosis due to AD or Psychosis in patientswith AD, apathy due to AD or apathy in patients with AD, psychiatricsymptoms of patients with Lewy body dementia, Progressive SupranuclearPalsy (PSP), Frontotemporal dementia (FTD or variants thereof), TBI(traumatic brain injury, acute or chronic), Corticobasal Degeneration(CBD), and Picks Disease. In particular, the tauopathies may be selectedfrom Alzheimer's disease, Argyrophilic Grain Disease (AGD), Psychosisdue to AD or Psychosis in patients with AD, apathy due to AD or apathyin patients with AD, and psychiatric symptoms of patients with Lewy bodydementia.

The treatment envisioned by the present invention may be chronic and thepatient may be treated at least 2 weeks, such as at least for 1 month,6, months, 1 year or more.

The antibodies of the present invention may, for example, be monoclonalantibodies produced by the hybridoma method first described by Kohler etal., Nature 256, 495 (1975), or may be monoclonal antibodies produced byrecombinant DNA or other methods, or more preferably may be produced bythe novel method disclosed. Monoclonal antibodies may also be isolatedfrom antibody phage display libraries using the techniques described in,for example, Clackson et al., Nature 352, 624-628 (1991) and Marks etal., J. Mol. Biol. 222, 581-597 (1991). Monoclonal antibodies may beobtained from any suitable source. Thus, for example, monoclonalantibodies may be obtained from hybridomas prepared from murine splenicB lymphocyte cells obtained from mice immunized with an antigen ofinterest, for instance, in the form of cells expressing the antigen onthe surface, or a nucleic acid encoding an antigen of interest.Monoclonal antibodies may also be obtained from hybridomas derived fromantibody-expressing cells of immunized humans or from non-human mammalssuch as rats, rabbits, dogs, sheep, goats, primates, etc.

In one embodiment, the antibody of the invention is a human antibody.Human monoclonal antibodies directed against tau may be generated usingtransgenic or trans-chromosomal mice carrying parts of the human immunesystem rather than the mouse system. Such transgenic andtranschromosomic mice include mice referred to herein as HuMAb (Humanmonoclonal antibody) mice and KM mice, respectively, and arecollectively referred to herein as “transgenic mice”.

The HuMAb mouse contains a human immunoglobulin gene mini-locus thatencodes un-rearranged human heavy variable and constant (p and Y) andlight variable and constant (K) chain immunoglobulin sequences, togetherwith targeted mutations that inactivate the endogenous p and K chainloci (Lonberg, N. et al., Nature 368, 856-859 (1994)). Accordingly, themice exhibit reduced expression of mouse IgM or K and in response toimmunization, the introduced human heavy and light chain transgenes,undergo class switching and somatic mutation to generate high affinityhuman IgG, κ monoclonal antibodies (Lonberg, N. et al. (1994), supra;reviewed in Lonberg, N., Handbook of Experimental Pharmacology 113,49-101 (1994), Lonberg, N. and Huszar, D., Intern. Rev. Immunol. Vol. 1365-93 (1995) and Harding, F. and Lonberg, N., Ann. N. Y. Acad. Sci 764536-546 (1995)). The preparation of HuMAb mice is described in detail inTaylor, L. et al., Nucleic Acids Research 20, 6287-6295 (1992), Chen, J.et al., International Immunology 5, 647-656 (1993), Tuaillon et al., J.Immunol. 152, 2912-2920 (1994), Taylor, L. et al., InternationalImmunology 6, 579-591 (1994), Fishwild, D. et al., Nature Biotechnology14, 845-851 (1996). See also U.S. Pat. No. 5,545,806, U.S. Pat. No.5,569,825, U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,633,425, U.S. Pat.No. 5,789,650, U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,661,016, U.S.Pat. No. 5,814,318, U.S. Pat. No. 5,874,299, U.S. Pat. No. 5,770,429,U.S. Pat. No. 5,545,807, WO 98/24884, WO 94/25585, WO 93/1227, WO92/22645, WO 92/03918 and WO 01/09187.

The HCo7, HCo12, HCo17 and HCo20 mice have a JKD disruption in theirendogenous light chain (kappa) genes (as described in Chen et al., EMBOJ. 12, 811-820 (1993)), a CMD disruption in their endogenous heavy chaingenes (as described in Example 1 of WO 01/14424), and a KCo5 human kappalight chain transgene (as described in Fishwild et al., NatureBiotechnology 14, 845-851 (1996)). Additionally, the HCo7 mice have aHCo7 human heavy chain transgene (as described in U.S. Pat. No.5,770,429), the HCo12 mice have a HCo12 human heavy chain transgene (asdescribed in Example 2 of WO 01/14424), the HCo17 mice have a HCo17human heavy chain transgene (as described in Example 2 of WO 01/09187)and the HCo20 mice have a HCo20 human heavy chain transgene. Theresulting mice express human immunoglobulin heavy and kappa light chaintransgenes in a background homozygous for disruption of the endogenousmouse heavy and kappa light chain loci.

In the KM mouse strain, the endogenous mouse kappa light chain gene hasbeen homozygously disrupted as described in Chen et al., EMBO J. 12,811-820 (1993) and the endogenous mouse heavy chain gene has beenhomozygously disrupted as described in Example 1 of WO 01/09187. Thismouse strain carries a human kappa light chain transgene, KCo5, asdescribed in Fishwild et al., Nature Biotechnology 14, 845-851 (1996).This mouse strain also carries a human heavy chain transchromosomecomposed of chromosome 14 fragment hCF (SC20) as described in WO02/43478. HCo12-Balb/c, HCo17-Balb/c and HCo20-Balb/c mice can begenerated by crossing HCo12, HCo17 and HCo20 to KCo5[J/K](Balb) asdescribed in WO 09/097006.

The rTg4510 mouse is a known tauopathy model providing temporal andspatial control over mutant tau transgene expression. In the KM mousestrain, the endogenous mouse kappa light chain gene has beenhomozygously disrupted as described in Chen et al., EMBO J. 12, 811-820(1993) and the endogenous mouse heavy chain gene has been homozygouslydisrupted as described in Example 1 of WO 01/09187. This mouse straincarries a human kappa light chain transgene, KCo5, as described inFishwild et al., Nature Biotechnology 14, 845-851 (1996). This mousestrain also carries a human heavy chain trans-chromosome composed ofchromosome 14 epitope-binding fragment hCF (SC20) as described in WO02/43478.

Splenocytes from these transgenic mice may be used to generatehybridomas that secrete human monoclonal antibodies according towell-known techniques. Human monoclonal or polyclonal antibodies of thepresent invention, or antibodies, of the present invention originatingfrom other species may also be generated transgenically through thegeneration of another non-human mammal or plant that is transgenic forthe immunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies may beproduced in, and recovered from, the milk of goats, cows, or othermammals (see for instance U.S. Pat. No. 5,827,690; U.S. Pat. No.5,756,687; U.S. Pat. No. 5,750,172 and U.S. Pat. No. 5,741,957).

The antibody of the invention may be of any isotype. The choice ofisotype typically will be guided by the desired effector functions, suchas ADCC induction. Exemplary isotypes are IgG1, IgG2, IgG3, and IgG4.Either of the human light chain constant domains, kappa or lambda, maybe used. If desired, the class of an anti-tau antibody of the presentinvention may be switched by known methods. For example, an antibody ofthe present invention that was originally IgM may be class switched toan IgG antibody of the present invention. Further, class switchingtechniques may be used to convert one IgG subclass to another, forinstance from IgG1 to IgG2. Thus, the effector function of theantibodies of the present invention may be changed by isotype switchingto, e.g., an IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibody forvarious therapeutic uses. In one embodiment an antibody of the presentinvention is an IgG1 antibody, for instance an IgG1, κ. An antibody issaid to be of a particular isotype if its amino acid sequence is mosthomologous to that isotype, relative to other isotypes.

In one embodiment, the antibody of the invention is a full-lengthantibody, preferably an IgG antibody, in particular an IgG1, κ antibody.In another embodiment, the antibody of the invention is an antibodyepitope-binding fragment or a single-chain antibody.

Antibodies and epitope-binding fragments thereof may e.g. be obtained byepitope-binding fragmentation using conventional techniques, andepitope-binding fragments screened for utility in the same manner asdescribed herein for whole antibodies. For example, F(ab′)₂epitope-binding fragments may be generated by treating antibody withpepsin. The resulting F(ab′)₂ epitope-binding fragment may be treated toreduce disulfide bridges to produce Fab′ epitope-binding fragments. Fabepitope-binding fragments may be obtained by treating an IgG antibodywith papain; Fab′ epitope-binding fragments may be obtained with pepsindigestion of IgG antibody. An F(ab′) epitope-binding fragment may alsobe produced by binding Fab′-described below via a thioether bond or adisulfide bond. A Fab′ epitope-binding fragment is an antibodyepitope-binding fragment obtained by cutting a disulfide bond of thehinge domain of the F(ab′)₂. A Fab′-epitope-binding fragment may beobtained by treating an F(ab′)2 epitope-binding fragment with a reducingagent, such as dithiothreitol. Antibody epitope-binding fragment mayalso be generated by expression of nucleic acids encoding suchepitope-binding fragments in recombinant cells (see for instance Evanset al., J. Immunol. Meth. 184, 123-38 (1995)). For example, a chimericgene encoding a portion of an F(ab′)2 epitope-binding fragment couldinclude DNA sequences encoding the CH1 domain and hinge domain of the Hchain, followed by a translational stop codon to yield such a truncatedantibody epitope-binding fragment molecule.

In one embodiment, the anti-tau antibody is a monovalent antibody,preferably a monovalent antibody as described in WO2007059782 (which isincorporated herein by reference in its entirety) having a deletion ofthe hinge region. Accordingly, in one embodiment, the antibody is amonovalent antibody, wherein said anti-tau antibody is constructed by amethod comprising: i) providing a nucleic acid construct encoding thelight chain of said monovalent antibody, said construct comprising anucleotide sequence encoding the VL region of a selected antigenspecific anti-tau antibody and a nucleotide sequence encoding theconstant CL region of an Ig, wherein said nucleotide sequence encodingthe VL region of a selected antigen specific antibody and saidnucleotide sequence encoding the CL region of an Ig are operably linkedtogether, and wherein, in case of an IgG1 subtype, the nucleotidesequence encoding the CL region has been modified such that the CLregion does not contain any amino acids capable of forming disulfidebonds or covalent bonds with other peptides comprising an identicalamino acid sequence of the CL region in the presence of polyclonal humanIgG or when administered to an animal or human being; ii) providing anucleic acid construct encoding the heavy chain of said monovalentantibody, said construct comprising a nucleotide sequence encoding theVH region of a selected antigen specific antibody and a nucleotidesequence encoding a constant CH region of a human Ig, wherein thenucleotide sequence encoding the CH region has been modified such thatthe region corresponding to the hinge region and, as required by the Igsubtype, other regions of the CH region, such as the CH3 region, doesnot comprise any amino acid residues which participate in the formationof disulphide bonds or covalent or stable non-covalent inter-heavy chainbonds with other peptides comprising an identical amino acid sequence ofthe CH region of the human Ig in the presence of polyclonal human IgG orwhen administered to an animal human being, wherein said nucleotidesequence encoding the VH region of a selected antigen specific antibodyand said nucleotide sequence encoding the CH region of said Ig areoperably linked together; iii) providing a cell expression system forproducing said monovalent antibody; iv) producing said monovalentantibody by co-expressing the nucleic acid constructs of (i) and (ii) incells of the cell expression system of (iii).

Similarly, in one embodiment, the anti-tau antibody of the invention isa monovalent antibody, which comprises:

-   -   (i) a variable domain of an antibody of the invention as        described herein or an epitope-binding part of the said domain,        and    -   (ii) a CH domain of an immunoglobulin or a domain thereof        comprising the CH2 and CH3 domains, wherein the CH domain or        domain thereof has been modified such that the domain        corresponding to the hinge domain and, if the immunoglobulin is        not an IgG4 subtype, other domains of the CH domain, such as the        CH3 domain, do not comprise any amino acid residues, which are        capable of forming disulfide bonds with an identical CH domain        or other covalent or stable non-covalent inter-heavy chain bonds        with an identical CH domain in the presence of polyclonal human        IgG.

In a further embodiment, the heavy chain of the monovalent antibody ofthe invention has been modified such that the entire hinge region hasbeen deleted.

In another further embodiment, the sequence of the monovalent antibodyhas been modified so that it does not comprise any acceptor sites forN-linked glycosylation.

The invention also includes “Bispecific Antibodies,” wherein an anti-taubinding region (e.g., a tau-binding region of an anti-tau monoclonalantibody) is part of a bivalent or polyvalent bispecific scaffold thattargets more than one epitope, (for example a second epitope couldcomprise an epitope of an active transport receptor, such that theBispecific Antibody would exhibit improved transcytosis across abiological barrier, such as the Blood Brain Barrier). Thus, in anotherfurther embodiment, the monovalent Fab of an anti-tau antibody may bejoined to an additional Fab or scfv that targets a different protein togenerate a bispecific antibody. A bispecific antibody can have a dualfunction, for example a therapeutic function imparted by an anti-taubinding domain and a transport function that can bind to a receptormolecule to enhance transfer cross a biological barrier, such as theblood brain barrier.

Antibodies and epitope-binding fragments thereof of the invention, alsoinclude single chain antibodies. Single chain antibodies are peptides inwhich the heavy and light chain Fv domains are connected. In oneembodiment, the present invention provides a single-chain Fv (scFv)wherein the heavy and light chains in the Fv of an anti-tau antibody ofthe present invention are joined with a flexible peptide linker(typically of about 10, 12, 15 or more amino acid residues) in a singlepeptide chain. Methods of producing such antibodies are described in forinstance U.S. Pat. No. 4,946,778, Pluckthun in The Pharmacology ofMonoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994), Bird et al., Science 242,423-426 (1988), Huston et al., PNAS USA 85, 5879-5883 (1988) andMcCafferty et al., Nature 348, 552-554 (1990). The single chain antibodymay be monovalent, if only a single VH and VL are used, bivalent, if twoVH and VL are used, or polyvalent, if more than two VH and VL are used.

The antibodies and epitope-binding fragments thereof described hereinmay be modified by inclusion of any suitable number of modified aminoacids and/or associations with such conjugated substituents. Suitabilityin this context is generally determined by the ability to at leastsubstantially retain the tau selectivity and/or the tau specificityassociated with the non-derivatized parent anti-tau antibody. Theinclusion of one or more modified amino acids may be advantageous in,for example, increasing polypeptide serum half-life, reducingpolypeptide antigenicity, or increasing polypeptide storage stability.Amino acid(s) are modified, for example, co-translationally orpost-translationally during recombinant production (e.g., N-linkedglycosylation at N-X-S/T motifs during expression in mammalian cells) ormodified by synthetic means. Non-limiting examples of a modified aminoacid include a glycosylated amino acid, a sulfated amino acid, aprenylated (e.g., farnesylated, geranyl-geranylated) amino acid, anacetylated amino acid, an acylated amino acid, a PEGylated amino acid, abiotinylated amino acid, a carboxylated amino acid, a phosphorylatedamino acid, and the like. References adequate to guide one of skill inthe modification of amino acids are replete throughout the literature.Example protocols are found in Walker (1998) Protein Protocols OnCD-Rom, Humana Press, Totowa, N.J. The modified amino acid may, forinstance, be selected from a glycosylated amino acid, a PEGylated aminoacid, a farnesylated amino acid, an acetylated amino acid, abiotinylated amino acid, an amino acid conjugated to a lipid moiety, oran amino acid conjugated to an organic derivatizing agent.

The antibodies and epitope-binding fragments thereof of the invention,may also be chemically modified by covalent conjugation to a polymer tofor instance increase their circulating half-life. Exemplary polymers,and methods to attach them to peptides, are illustrated in for instanceU.S. Pat. No. 4,766,106; U.S. Pat. No. 4,179,337; U.S. Pat. No.4,495,285 and U.S. Pat. No. 4,609,546. Additional illustrative polymersinclude polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., aPEG with a molecular weight of between about 1,000 and about 40,000,such as between about 2,000 and about 20,000, e.g., about 3,000-12,000g/mol).

The antibodies and epitope-binding fragments thereof of the presentinvention may further be used in a diagnostic method or as a diagnosticimaging ligand.

In one embodiment, antibodies and epitope-binding fragments thereof ofthe invention comprising one or more radiolabeled amino acids areprovided. A radiolabeled anti-tau antibody may be used for bothdiagnostic and therapeutic purposes (conjugation to radiolabeledmolecules is another possible feature). Non-limiting examples of suchlabels include, but are not limited to bismuth (²¹³Bi), carbon (¹¹C,¹³C, ¹⁴C), chromium (⁵¹Cr), cobalt (⁵⁷Co, ⁶⁰Co), copper (⁶⁴Cu),dysprosium (¹⁶⁵Dy), erbium (¹⁶⁹Er), fluorine (¹⁸F), gadolinium (¹⁵³Gd,¹⁵⁹Gd), gallium (⁶⁸Ga, ⁶⁷Ga), germanium (⁶⁸Ge), gold (¹⁹⁸Au), holmium(¹⁶⁶Ho), hydrogen (³H), indium (¹¹¹In, ¹¹²In, ¹¹³In, ¹¹⁵In) iodine(¹²¹I, ¹²³I, ¹²⁵I, ¹³¹I), iridium (¹⁹²Ir), iron (⁵⁹Fe), krypton(^(81m)Kr), lanthanium (¹⁴⁰La), lutelium (¹⁷⁷Lu), manganese (⁵⁴Mn),molybdenum (⁹⁹Mo), nitrogen (¹³N, ¹⁵N), oxygen (¹⁵O), palladium (¹⁰³Pd),phosphorus (³²P), potassium (⁴²K), praseodymium (¹⁴²Pr), promethium(¹⁴⁹Pm), rhenium (¹⁸⁶Re, ¹⁸⁸Re), rhodium (¹⁰⁵Rh), rubidium (⁸¹Rb, ⁸²Rb),ruthenium (⁸²Ru, ⁹⁷Ru), samarium (¹⁵³Sm), scandium (⁴⁷Sc), selenium(⁷⁵Se), sodium (²⁴Na), strontium (⁸⁵Sr, ⁸⁹Sr, ⁹²Sr), sulfur (³⁵S),technetium (⁹⁹Tc), thallium (²⁰¹Tl), tin (¹¹³Sn, ¹¹⁷Sn), xenon (¹³³Xe),ytterbium (¹⁶⁹Yb, ¹⁷⁵Yb, ¹⁷⁷Yb), yttrium (⁹⁰Y), zinc (⁶⁵Zn) andzirconium (⁸⁹Zr). Zirconium (⁸⁹Zr) is particularly interesting. Methodsfor preparing radiolabeled amino acids and related peptide derivativesare known in the art (see for instance Junghans et al., in CancerChemotherapy and Biotherapy 655-686 (2nd edition, Chafner and Longo,eds., Lippincott Raven (1996)) and U.S. Pat. No. 4,681,581; U.S. Pat.No. 4,735,210; U.S. Pat. No. 5,101,827; U.S. Pat. No. 5,102,990 (U.S.RE35,500), U.S. Pat. No. 5,648,471 and U.S. Pat. No. 5,697,902. Forexample, a radioisotope may be conjugated by a chloramine T method(Lindegren, S. et al. (1998) “Chloramine-T In High-Specific-ActivityRadioiodination Of Antibodies UsingN-Succinimidyl-3-(Trimethylstannyl)Benzoate As An Intermediate,” Nucl.Med. Biol. 25(7):659-665; Kurth, M. et al. (1993) “Site-SpecificConjugation Of A Radioiodinated Phenethylamine Derivative To AMonoclonal Antibody Results In Increased Radioactivity Localization InTumor,” J. Med. Chem. 36(9):1255-1261; Rea, D. W. et al. (1990)“Site-specifically radioiodinated antibody for targeting tumors,” CancerRes. 50(3 Suppl):857s-861s).

The invention also provides anti-tau antibodies and epitope-bindingfragments thereof that are detectably labeled using a fluorescent label(such as a rare earth chelate (e.g., a europium chelate)), afluorescein-type label (e.g., fluorescein, fluorescein isothiocyanate,5-carboxyfluorescein, 6-carboxy fluorescein, dichlorotriazinylaminefluorescein), a rhodamine-type label (e.g., ALEXA FLUOR® 568(Invitrogen), TAMRA® or dansyl chloride), VIVOTAG 680 XL FLUOROCHROME™(Perkin Elmer), phycoerythrin; umbelliferone, Lissamine; a cyanine; aphycoerythrin, Texas Red, BODIPY FL-SE® (Invitrogen) or an analoguethereof, all of which are suitable for optical detection.Chemiluminescent labels may be employed (e.g., luminol, luciferase,luciferin, and aequorin). Such diagnosis and detection can also beaccomplished by coupling the diagnostic molecule of the presentinvention to detectable substances including, but not limited to,various enzymes, enzymes including, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase, or to prosthetic group complexes such as, but notlimited to, streptavidin/biotin and avidin/biotin.

Chemiluminescent labels may be employed (e.g., luminol, luciferase,luciferin, and aequorin). Such diagnosis and detection can also beaccomplished by coupling the diagnostic molecule of the presentinvention to detectable substances including, but not limited to,various enzymes, enzymes including, but not limited to, horseradishperoxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase, or to prosthetic group complexes such as, but notlimited to, streptavidin/biotin and avidin/biotin. Paramagnetic labelscan also be employed, and are preferably detected using PositronEmission Tomography (PET) or Single-Photon Emission Computed Tomography(SPECT). Such paramagnetic labels include, but are not limited tocompounds containing paramagnetic ions of Aluminum (Al), Barium (Ba),Calcium (Ca), Cerium (Ce), Dysprosium (Dy), Erbium (Er), Europium (Eu),Gandolinium (Gd), Holmium (Ho), Iridium (Ir), Lithium (Li), Magnesium(Mg), Manganese (Mn), Molybdenum (M), Neodymium (Nd), Osmium (Os),Oxygen (O), Palladium (Pd), Platinum (Pt), Rhodium (Rh), Ruthenium (Ru),Samarium (Sm), Sodium (Na), Strontium (Sr), Terbium (Tb), Thulium (Tm),Tin (Sn), Titanium (Ti), Tungsten (W), and Zirconium (Zi), andparticularly, Co⁺², CR⁺², Cr⁺³, Cu⁺², Fe⁺², Fe⁺³, Ga⁺³, Mn⁺³, Ni⁺²,Ti⁺³, V⁺³, and V⁺⁴, positron emitting metals using various positronemission tomographies, and non-radioactive paramagnetic metal ions.

Thus in one embodiment the anti-tau antibody or tau-binding fragmentthereof of the invention may be labelled with a fluorescent label, achemiluminescent label, a paramagnetic label, a radioisotopic label oran enzyme label. The labelled antibody of fragment may be used indetecting or measuring the presence or amount of said tau in the brainof a subject. This method may comprise the detection or measurement ofin vivo imaging of anti-tau antibody or tau-binding fragment bound tosaid tau and may comprises ex vivo imaging of said anti-tau antibody ortau-binding fragment bound to such tau.

In a further aspect, the invention relates to an expression vectorencoding one or more polypeptide chains of an antibody of the inventionor a tau-binding fragment thereof. Such expression vectors may be usedfor recombinant production of antibodies or epitope-binding fragmentsthereof of the invention.

An expression vector in the context of the present invention may be anysuitable DNA or RNA vector, including chromosomal, non-chromosomal, andsynthetic nucleic acid vectors (a nucleic acid sequence comprising asuitable set of expression control elements). Examples of such vectorsinclude derivatives of SV40, bacterial plasmids, phage DNA, baculovirus,yeast plasmids, vectors derived from combinations of plasmids and phageDNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, ananti-tau antibody-encoding nucleic acid is comprised in a naked DNA orRNA vector, including, for example, a linear expression element (asdescribed in, for instance, Sykes and Johnston, Nat Biotech 12, 355-59(1997)), a compacted nucleic acid vector (as described in for instanceU.S. Pat. No. 6,077,835 and/or WO 00/70087), a plasmid vector such aspBR322, pUC 19/18, or pUC 118/119, a “midge” minimally-sized nucleicacid vector (as described in, for instance, Schakowski et al., Mol Ther3, 793-800 (2001)), or as a precipitated nucleic acid vector construct,such as a CaPO₄-precipitated construct (as described in, for instance,WO 00/46147, Benvenisty and Reshef, PNAS USA 83, 9551-55 (1986), Wigleret al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic CellGenetics 2, 603 (1981)). Such nucleic acid vectors and the usage thereofare well known in the art (see for instance U.S. Pat. No. 5,589,466 andU.S. Pat. No. 5,973,972).

In one embodiment, the vector is suitable for expression of anti-tauantibodies or epitope-binding fragments thereof of the invention in abacterial cell. Examples of such vectors include expression vectors suchas BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J. Biol.Chem. 264, 5503-5509 (1989), pET vectors (Novagen, Madison, Wis.), andthe like.

An expression vector may also or alternatively be a vector suitable forexpression in a yeast system. Any vector suitable for expression in ayeast system may be employed. Suitable vectors include, for example,vectors comprising constitutive or inducible promoters such as alphafactor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed.Current Protocols in Molecular Biology, Greene Publishing and WleyInterScience New York (1987), Grant et al., Methods in Enzymol 153,516-544 (1987), Mattanovich, D. et al. Methods Mol. Biol. 824, 329-358(2012), Celik, E. et al. Biotechnol. Adv. 30(5), 1108-1118 (2012), Li,P. et al. Appl. Biochem. Biotechnol. 142(2), 105-124 (2007), Böer, E. etal. Appl. Microbiol. Biotechnol. 77(3), 513-523 (2007), van der Vaart,J. M. Methods Mol. Biol. 178, 359-366 (2002), and Holliger, P. MethodsMol. Biol. 178, 349-357 (2002)).

In an expression vector of the invention, anti-tau antibody-encodingnucleic acids may comprise or be associated with any suitable promoter,enhancer, and other expression-facilitating elements. Examples of suchelements include strong expression promoters (e.g., human CMV IEpromoter/enhancer as well as RSV, SV40, SL3-3, MMTV, and HIV LTRpromoters), effective poly (A) termination sequences, an origin ofreplication for plasmid product in E. coli, an antibiotic resistancegene as selectable marker, and/or a convenient cloning site (e.g., apolylinker). Nucleic acids may also comprise an inducible promoter asopposed to a constitutive promoter such as CMV IE (the skilled artisanwill recognize that such terms are actually descriptors of a degree ofgene expression under certain conditions).

In an even further aspect, the invention relates to a recombinanteukaryotic or prokaryotic host cell, such as a transfectoma, whichproduces an antibody or epitope-binding fragment thereof of theinvention as defined herein or a bispecific molecule of the invention asdefined herein. Examples of host cells include yeast, bacteria, andmammalian cells, such as CHO or HEK cells. For example, in oneembodiment, the present invention provides a cell comprising a nucleicacid stably integrated into the cellular genome that comprises asequence coding for expression of an anti-tau antibody of the presentinvention or an epitope-binding fragment thereof. In another embodiment,the present invention provides a cell comprising a non-integratednucleic acid, such as a plasmid, cosmid, phagemid, or linear expressionelement, which comprises a sequence coding for expression of an anti-tauantibody or epitope-binding fragment thereof of the invention.

In a further aspect, the invention relates to a method for producing ananti-tau antibody of the invention, said method comprising the steps ofa) culturing a hybridoma or a host cell of the invention as describedherein above, and b) purifying the antibody of the invention from theculture media.

In one embodiment, the invention relates to a preparation that, as suchterm is used herein, comprises an anti-tau antibody as defined herein,and that is substantially free of naturally-arising antibodies that areeither not capable of binding to tau or that do not materially alter theanti-tau functionality of the preparation. Thus, such a preparation doesnot encompass naturally-arising serum, or a purified derivative of suchserum, that comprises a mixture of an anti-tau antibody and anotherantibody that does not alter the functionality of the anti-tau antibodyof the preparation, wherein such functionality is:

-   -   (i) a substantial inability to bind to non-phosphorylated tau;    -   (ii) a substantial inability to bind to tau that is        phosphorylated at S404 and not phosphorylated at S396;    -   (iii) the ability to bind to tau phosphorylated at S396;    -   (iv) the ability to bind to tau phosphorylated at both S396 and        at S404;    -   (v) the ability to selectively discriminate between        phosphorylated tau residues S396 and S404 such that it is        substantially unable to bind the phosphorylated 404 residue;    -   (vi) the ability to bind hyper-phosphorylated tau from human        Alzheimer's disease brains;    -   (vii) the ability to discriminate between pathological and        non-pathological human tau protein; and/or    -   (viii) the capability, when used as described herein with        immune-depleted rTg4510 extracts from transgenic mice, to        specifically reduce the hyperphosphorylated tau 64 kDa and 70        kDa bands by at least 90%, while not reducing the 55 kDa tau        band by more than 10% or the capability, when used as described        herein with extracts from human AD post-mortem brains, to        specifically reduce the S396 phosphorylated hyperphosphorylated        tau bands by at least 90%, while not reducing the        non-hyperphosphorylated tau bands by more than 10%.

The invention particularly relates to preparations of such an anti-tauantibody having a structural change in its amino acid sequence (in anyof its CDRs, variable domains, framework residues and/or constantdomains) relative to the structure of a naturally-occurring anti-tauantibody, wherein said structural change causes the anti-tau antibody toexhibit a markedly altered functionality (i.e., more than a 20%difference, more than a 40% difference, more than a 60% difference, morethan an 80% difference, more than a 100% difference, more than a 150%difference, more than a 2-fold difference, more than a 4-folddifference, more than a 5-fold difference, or more than a 10-folddifference in functionality) relative to the functionality exhibited bysaid naturally-occurring anti-tau antibody; wherein such functionalityis:

-   -   (i) a substantial inability to bind to non-phosphorylated tau;    -   (ii) a substantial inability to bind to tau that is        phosphorylated at S404 and not phosphorylated at S396;    -   (iii) the ability to bind to tau phosphorylated at S396;    -   (iv) the ability to bind to tau phosphorylated at both S396 and        at S404;    -   (v) the ability to selectively discriminate between        phosphorylated tau residues S396 and S404 such that it is        substantially unable to bind the phosphorylated 404 residue;    -   (vi) the ability to bind hyper-phosphorylated tau from human        Alzheimer's disease brains;    -   (vii) the ability to discriminate between pathological and        non-pathological human tau protein; and/or    -   (viii) the capability, when used as described herein with        immune-depleted rTg4510 extracts from transgenic mice, to        specifically reduce the hyperphosphorylated tau 64 kDa and 70        kDa bands by at least 90%, while not reducing the 55 kDa tau        band by more than 10%; or the capability, when used as described        herein with extracts from human AD post-mortem brains to        specifically reduce the S396 phosphorylated hyperphosphorylated        tau bands by at least 90%, while not reducing the        non-hyperphosphorylated tau bands by more than 10%.

The term “substantially free” of naturally-arising antibodies refers tothe complete absence of such naturally-arising antibodies in suchpreparations, or of the inclusion of a concentration of suchnaturally-arising antibodies in such preparations that does notmaterially affect the tau-binding properties of the preparations. Anantibody is said to be “isolated” if it has no naturally-arisingcounterpart or has been separated or purified from components whichnaturally accompany it.

The term “naturally-arising antibodies,” as it relates to suchpreparations, refers to antibodies (including naturally-arisingautoantibodies) elicited within living humans or other animals, as anatural consequence to the functioning of their immune systems.

Thus, the preparations of the present invention do not exclude, andindeed explicitly encompass, such preparations that contain an anti-tauantibody and a deliberately added additional antibody capable of bindingto an epitope that is not possessed by tau. Such preparationsparticularly include embodiments thereof wherein the preparationexhibits enhanced efficacy in treating Alzheimer's disease (AD),Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP),and Corticobasal Degeneration (CBD). Furthermore, the present inventionis directed to preparations that contain an anti-tau antibodyantibodies, or epitope-binding fragments thereof, intended for use inthe treatment of Psychosis, particularly Psychosis due to AD orPsychosis in patients with AD, apathy due to AD or apathy in patientswith AD, and psychiatric symptoms of patients with Lewy body dementia.Furthermore, the preparations of the present invention contain ananti-tau antibody antibodies, or epitope-binding fragments thereof, thatmay be used in the treatment of stroke, stroke recovery,neurodegeneration in relation to Parkinson's disease.

In an even further aspect, the invention relates to a pharmaceuticalcomposition comprising:

-   -   (i) a tau antibody, or epitope-binding fragment thereof, both as        defined herein, or a preparation, as such term is defined        herein, that comprises such an anti-tau antibody or        epitope-binding fragment thereof; and    -   (ii) a pharmaceutically-acceptable carrier.

The pharmaceutical compositions may be formulated with pharmaceuticallyacceptable carriers or diluents as well as any other known adjuvants andexcipients in accordance with conventional techniques such as thosedisclosed in Remington: The Science and Practice of Pharmacy, 22ndEdition, Gennaro, Ed., Mack Publishing Co., Easton, Pa., 2013.

The pharmaceutically acceptable carriers or diluents as well as anyother known adjuvants and excipients should be suitable for the chosencompound of the present invention and the chosen mode of administration.Suitability for carriers and other components of pharmaceuticalcompositions is determined based on the lack of significant negativeimpact on the desired biological properties of the chosen compound orpharmaceutical composition of the present invention (e.g., less than asubstantial impact (10% or less relative inhibition, 5% or less relativeinhibition, etc.)) on epitope binding.

A pharmaceutical composition of the present invention may also includediluents, fillers, salts, buffers, detergents (e.g., a nonionicdetergent, such as Tween-20 or Tween-80), stabilizers (e.g., sugars orprotein-free amino acids), preservatives, tissue fixatives,solubilizers, and/or other materials suitable for inclusion in apharmaceutical composition. The diluent is selected to not to affect thebiological activity of the combination. Examples of such diluents aredistilled water, physiological phosphate-buffered saline, Ringer'ssolutions, dextrose solution, and Hank's solution. In addition, thepharmaceutical composition or formulation may also include othercarriers, or non-toxic, nontherapeutic, non-immunogenic stabilizers andthe like. The compositions may also include large, slowly metabolizedmacromolecules, such as proteins, polysaccharides like chitosan,polylactic acids, polyglycolic acids and copolymers (e.g., latexfunctionalized sepharose, agarose, cellulose, and the like), polymericamino acids, amino acid copolymers, and lipid aggregates (e.g., oildroplets or liposomes).

The actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present invention may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration. The selected dosage level will depend upon avariety of pharmacokinetic factors including the activity of theparticular compositions of the present invention employed, or the amidethereof, the route of administration, the time of administration, therate of excretion of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compositions employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

The pharmaceutical composition may be administered by any suitable routeand mode, including: parenteral, topical, oral or intranasal means forprophylactic and/or therapeutic treatment. In one embodiment, apharmaceutical composition of the present invention is administeredparenterally. The phrases “parenteral administration” and “administeredparenterally” as used herein means modes of administration other thanenteral and topical administration, usually by injection, and includeepidermal, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,intratendinous, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intracranial,intrathoracic, epidural and intrasternal injection and infusion.

Additional suitable routes of administering a compound of the presentinvention in vivo and in vitro are well known in the art and may beselected by those of ordinary skill in the art.

In one embodiment that pharmaceutical composition is administered byintravenous or subcutaneous injection or infusion.

Pharmaceutically acceptable carriers include any and all suitablesolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonicity agents, antioxidants and absorption delaying agents,and the like that are physiologically compatible with a compound of thepresent invention.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the present inventioninclude water, saline, phosphate buffered saline, ethanol, dextrose,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloidal solutions, tragacanth gum and injectable organicesters, such as ethyl oleate, and/or various buffers. Other carriers arewell known in the pharmaceutical arts.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe present invention is contemplated.

Proper fluidity may be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

Pharmaceutical compositions of the present invention may also comprisepharmaceutically acceptable antioxidants for instance (1) water solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfite, sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like.

Pharmaceutical compositions of the present invention may also compriseisotonicity agents, such as sugars, polyalcohols, such as mannitol,sorbitol, glycerol or sodium chloride in the compositions.

The pharmaceutical compositions of the present invention may alsocontain one or more adjuvants appropriate for the chosen route ofadministration such as preservatives, wetting agents, emulsifyingagents, dispersing agents, preservatives or buffers, which may enhancethe shelf life or effectiveness of the pharmaceutical composition. Thecompounds of the present invention may be prepared with carriers thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Such carriers may include gelatin,glyceryl monostearate, glyceryl distearate, biodegradable, biocompatiblepolymers such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, collagen, polyorthoesters, and polylactic acid alone or with awax, or other materials well known in the art. Methods for thepreparation of such formulations are generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In one embodiment, the compounds of the present invention may beformulated to ensure proper distribution in vivo. Pharmaceuticallyacceptable carriers for parenteral administration include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. The use of such media and agents for pharmaceutically activesubstances is known in the art. Except insofar as any conventional mediaor agent is incompatible with the active compound, use thereof in thepharmaceutical compositions of the present invention is contemplated.Supplementary active compounds may also be incorporated into thecompositions.

Pharmaceutical compositions for injection must typically be sterile andstable under the conditions of manufacture and storage. The compositionmay be formulated as a solution, micro-emulsion, liposome, or otherordered structure suitable to high drug concentration. The carrier maybe an aqueous or non-aqueous solvent or dispersion medium containing forinstance water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. The proper fluidity may be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. In many cases, it will be preferable to include isotonicagents, for example, sugars, polyalcohols such as glycerol, mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe injectable compositions may be brought about by including in thecomposition an agent that delays antibody absorption, for example,monostearate salts and gelatin. Sterile injectable solutions may beprepared by incorporating the active compound in the required amount inan appropriate solvent with one or a combination of ingredients e.g. asenumerated above, as required, followed by sterilizationmicrofiltration. Generally, dispersions are prepared by incorporatingthe active compound into a sterile vehicle that contains a basicdispersion medium and the required other ingredients e.g. from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Dosage regimens in the above methods of treatment and uses describedherein are adjusted to provide the optimum desired response (e.g., atherapeutic response). For example, a single bolus may be administered,several divided doses may be administered over time or the dose may beproportionally reduced or increased as indicated by the exigencies ofthe therapeutic situation. Parenteral compositions may be formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe present invention are dictated by and directly dependent on (a) theunique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

The effective dosages and the dosage regimens for the antibodies orepitope-binding fragments thereof of the invention depend on the diseaseor condition to be treated and may be determined by persons skilled inthe art. On any given day that a dosage is given, the dosage may rangefrom about 0.0001 to about 100 mg/kg, and more usually from about 0.01to about 5 mg/kg, of the host body weight. For example, dosages can be 1mg/kg body weight or 10 mg/kg body weight or within the range of 1-10mg/kg body weight. Exemplary dosages thus include: from about 0.1 toabout 10 mg/kg/body weight, from about 0.1 to about 5 mg/kg/body weight,from about 0.1 to about 2 mg/kg/body weight, from about 0.1 to about 1mg/kg/body weight, for instance about 0.15 mg/kg/body weight, about 0.2mg/kg/body weight, about 0.5 mg/kg/body weight, about 1 mg/kg/bodyweight, about 1.5 mg/kg/body weight, about 2 mg/kg/body weight, about 5mg/kg/body weight, or about 10 mg/kg/body weight.

A physician having ordinary skill in the art may readily determine andprescribe the effective amount of the pharmaceutical compositionrequired. For example, the physician could start doses of an antibody orepitope-binding fragment thereof of the invention employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved. In general, a suitabledaily dose of a composition of the present invention will be that amountof the compound which is the lowest dose effective to produce atherapeutic effect. Such an effective dose will generally depend uponthe factors described above. Administration may e.g. be intravenous,intramuscular, intraperitoneal, or subcutaneous. If desired, theeffective daily dose of a pharmaceutical composition may be administeredas two, three, four, five, six or more sub-doses administered separatelyat appropriate intervals throughout the day, optionally, in unit dosageforms. While it is possible for a compound of the present invention tobe administered alone, it is preferable to administer the compound as apharmaceutical composition as described above.

The labeled antibodies or epitope-binding fragments thereof of theinvention can be used for diagnostic purposes to detect, diagnose, ormonitor diseases or disorders. The invention provides for the detectionor diagnosis of a neurodegenerative or cognitive disease or disorder,including but not limited to Alzheimer's disease, Argyrophilic GrainDisease (AGD), Progressive Supranuclear Palsy (PSP), and CorticobasalDegeneration (CBD), comprising: (a) assaying the existence ofpyroglutamated AR fragments in cells or tissue samples of a subjectusing one or more antibodies that specifically bind to tau; and (b)comparing the level of the antigen with a control level, e.g. levels innormal tissue samples, whereby an increase in the assayed level ofantigen compared to the control level of antigen is indicative of thedisease or disorder, or indicative of the severity of the disease ordisorder.

The antibodies or epitope-binding fragments thereof of the invention canbe used to assay tau or fragments of tau in a biological sample usingimmunohistochemical methods well-known in the art. Other antibody-basedmethods useful for detecting protein include immunoassays such as theenzyme linked immunoassay (ELISA) and the radioimmunoassay assay (RIA)and mesoscale discovery platform based assays (MSD). Suitable antibodylabels may be used in such kits and methods, and labels known in the artinclude enzyme labels, such as alkaline phosphatase and glucose oxidase;radioisotope labels, such as iodine (¹²⁵I, ¹³¹I)carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹²¹In), and technetium (^(99m)Tc); andluminescent labels, such as luminol and luciferase; and fluorescentlabels, such as fluorescein and rhodamine.

The presence of labeled anti-tau antibodies or their tau-bindingfragments may be detected in vivo for diagnostic purposes. In oneembodiment, diagnosis comprises: a) administering to a subject aneffective amount of such labeled molecule; b) waiting for a timeinterval following administration to allow the labeled molecule toconcentrate at sites (if any) of AR deposition and to allow for unboundlabeled molecule to be cleared to background level; c) determining abackground level; and d) detecting the labeled molecule in the subject,such that detection of labeled molecule above the background level isindicative that the subject has the disease or disorder, or isindicative of the severity of the disease or disorder. In accordancewith such embodiment, the molecule is labeled with an imaging moietysuitable for detection using a particular imaging system known to thoseskilled in the art. Background levels may be determined by variousmethods known in the art, including comparing the amount of labeledantibody detected to a standard value previously determined for aparticular imaging system. Methods and systems that may be used in thediagnostic methods of the invention include, but are not limited to,computed tomography (CT), whole body scan such as positron emissiontomography (PET), magnetic resonance imaging (MRI), and sonography.

In a further aspect, the invention provides a monoclonal antibody, or anepitope-binding fragment thereof, as defined herein for use in therapy.

In a further aspect, the invention provides a monoclonal antibody, or anepitope-binding fragment thereof, as defined herein for use in treating,diagnosing or imaging of tauopathies.

In a further aspect, the invention provides a monoclonal antibody, or anepitope-binding fragment thereof, as defined herein for use in treatingAlzheimer's disease, Argyrophilic Grain Disease (AGD), ProgressiveSupranuclear Palsy (PSP), and Corticobasal Degeneration (CBD).

In a further aspect, the invention provides a monoclonal antibody, or anepitope-binding fragment thereof, as defined herein for use in themanufacture of a medicament for treating, diagnosing or imagingtauopathies.

Preferably, the medicament is for treating Alzheimer's disease (AD),Argyrophilic Grain Disease (AGD), Progressive Supranuclear Palsy (PSP),and Corticobasal Degeneration (CBD) most preferably Alzheimer's disease(AD). The medicament is also preferably for the treatment of Psychosis,particularly Psychosis due to AD or Psychosis in patients with AD,apathy due to AD or apathy in patients with AD, and psychiatric symptomsof patients with Lewy body dementia.

In a further aspect, the invention provides a method of treating,diagnosing or imaging Alzheimer's disease or other tauopathies in asubject, said method comprising administering the medicament monoclonalantibody or epitope-binding fragment thereof as defined herein, to saidsubject in an effective amount.

In a preferred embodiment, the treatment is chronic, preferably for atleast 2 weeks, such as at least for 1 month, 6, months, 1 year or more.

In a further aspect, the invention provides a kit comprising theantibody, or fragment thereof, as defined herein for use in therapy.

List of Embodiments

The present invention particularly concerns the following embodiments:

-   -   1. A monoclonal antibody, or an epitope-binding fragment        thereof, capable of specifically binding to the phosphorylated        residue 396 of human tau (SEQ ID NO:1) such that the antibody or        an epitope-binding fragment thereof does not substantially bind        to SEQ ID NO:1 phosphorylated at residue 404 when residue 396 is        not phosphorylated.    -   2. The monoclonal antibody, or epitope-binding fragment thereof,        according to embodiment 1, which inhibits AD-P3 in the fluid        phase inhibition assay such that the monoclonal antibody, or        epitope-binding fragment thereof has an IC50 of 100 nM or less,        such as from 0.1 nM to 100 nM, such as at a concentration of 50        nM or less, such as from 0.1 nM to 50 nM.    -   3. The monoclonal antibody, or epitope-binding fragment thereof,        according to any one embodiments 1 or 2, wherein according to        Western Blot signal of pS396 Tau after immunodepletion studies        on Alzheimers diseased brain extracts capable of removing at        least 15% Tau phosphorylated at serine 396 from Alzheimer brain        homogenates at about 75 ng of antibody.    -   4. A monoclonal antibody, or epitope-binding fragment thereof,        comprising        -   (a) a Light Chain CDR1 comprising the amino acid sequence            selected from the group consisting of SEQ ID NO:3, SEQ ID            NO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34; SEQ ID            NO:35; SEQ ID NO:36; SEQ ID NO:37; SEQ ID NO:38; SEQ ID            NO:40; and SEQ ID NO:46;        -   (b) a Light Chain CDR2 comprising the amino acid sequence of            SEQ ID NO:4; SEQ ID NO:41; and SEQ ID NO:47;        -   (c) a Light Chain CDR3 comprising the amino acid sequence of            SEQ ID NO:5; SEQ ID NO:42; and SEQ ID NO:48;        -   (d) a Heavy Chain CDR1 comprising the amino acid sequence of            SEQ ID NO:6; SEQ ID NO:43; SEQ ID NO:49; SEQ ID NO:52; and            SEQ ID NO:55;        -   (e) a Heavy Chain CDR2 comprising the amino acid sequence            selected from the group consisting of SEQ ID NO:7; SEQ ID            NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:44; SEQ ID            NO:50; SEQ ID NO:53; and SEQ ID NO:56; and        -   (f) a Heavy Chain CDR3 comprising the amino acid sequence            selected from the group consisting of SEQ ID NO:8, SEQ ID            NO:39; SEQ ID NO:45; SEQ ID NO:51; SEQ ID NO:54; and SEQ ID            NO:57.    -   5. A monoclonal antibody, or an epitope-binding fragment        thereof, comprising        -   (a) a Light Chain selected from the group consisting of SEQ            ID NO:12; SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID            NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22 and SEQ ID            NO:23; and        -   (b) a Heavy Chain selected from the group consisting of SEQ            ID NO:11; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID            NO:24; SEQ ID NO:25; SEQ ID NO:26; and SEQ ID NO:27.    -   6. The monoclonal antibody, or an epitope-binding fragment        thereof, according to embodiment 1 or 5, wherein        -   (a) the Light Chain is SEQ ID NO:12; and        -   (b) the Heavy Chain is selected from the group consisting of            SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:24, SEQ            ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.    -   7. The monoclonal antibody, or an epitope-binding fragment        thereof, according to embodiment 1 or 5, wherein        -   (a) the Light Chain is selected from the group consisting of            SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:19; SEQ            ID NO:20; SEQ ID NO:21; SEQ ID NO:22 and SEQ ID NO:23; and        -   (b) the Heavy Chain is SEQ ID NO:11.    -   8. The monoclonal antibody, or epitope binding fragment thereof,        according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:28 and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   9. The monoclonal antibody, or epitope binding fragment thereof,        according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:12:        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:13.    -   10. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising at least one        of        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:28 and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   11. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:29; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   12. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:12; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:14.    -   13. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising at least one        of        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:29; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   14. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:30; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   15. The monoclonal antibody, or epitope binding fragment        thereof, according embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:12; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:15.    -   16. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising at least one        of        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:30; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   17. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:31;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   18. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:16; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:11.    -   19. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:31;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   20. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:32;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   21. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:17; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:11.    -   22. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:32;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8;    -   23. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:33;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   24. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:18; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:11.    -   25. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:33;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8;    -   26. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:34;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   27. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:19; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:11.    -   28. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:34;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   29. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:35;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   30. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:20; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:11.    -   31. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:35;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   32. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:36;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   33. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:21;        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:11.    -   34. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:36;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   35. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:37;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   36. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:22; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:11.    -   37. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:37;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   38. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:38;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   39. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:23; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:11.    -   40. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:38;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   41. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39.    -   42. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:12; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:24.    -   43. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising at least one        of        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39.    -   44. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:31;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39.    -   45. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:16;        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:24.    -   46. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:31; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39;        -   and further comprising at least one of        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6; and        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7.    -   47. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:32;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39.    -   48. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:17; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:24.    -   49. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:32; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39;        -   and further comprising at least one of        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6; and        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7.    -   50. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:31;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:28; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   51. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:16;        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:13.    -   52. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:31; and        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:28;        -   and further comprising at least one of        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   53. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:32;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:28; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   54. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:17;        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:13.    -   55. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:32; and        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:28;        -   and further comprising at least one of        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:8.    -   56. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:33;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39.    -   57. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:18; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:24.    -   58. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:33; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39;        -   and further comprising at least one of        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6; and        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7.    -   59. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:34;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39.    -   60. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:19; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:24.    -   61. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:34; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39;        -   and further comprising at least one of        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising at least one of        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:7.    -   62. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:28; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39.    -   63. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:12; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:25.    -   64. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:28; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39.    -   65. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:29; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39.    -   66. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:12; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:26.    -   67. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   and further comprising        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:29; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39.    -   68. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4;        -   (c) a Light Chain CDR3 having the amino acid sequence of SEQ            ID NO:5;        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:30; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39.    -   69. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 5, comprising        -   (a) a Light Chain comprising the amino acid sequence of SEQ            ID NO:12; and        -   (b) a Heavy Chain comprising the amino acid sequence of SEQ            ID NO:27.    -   70. The monoclonal antibody, or epitope binding fragment        thereof, according to embodiment 1 or 4, comprising        -   (a) a Light Chain CDR1 having the amino acid sequence of SEQ            ID NO:3;        -   (b) a Light Chain CDR2 having the amino acid sequence of SEQ            ID NO:4; and        -   (c) a Light Chain CDR3 having the amino acid sequence of            SEgQ ID NO:5;        -   and further comprising        -   (d) a Heavy Chain CDR1 having the amino acid sequence of SEQ            ID NO:6;        -   (e) a Heavy Chain CDR2 having the amino acid sequence of SEQ            ID NO:30; and        -   (f) a Heavy Chain CDR3 having the amino acid sequence of SEQ            ID NO:39.    -   71. The monoclonal antibody, or epitope-binding fragment        thereof, according to any one of embodiments 1 to 5, selective        for an amino acid motif of hyperphosphorylated tau whose motif        comprises of a phosphorylated serine residue and tyrosine        residue spaced by a single residue.    -   72. The monoclonal antibody or epitope-binding fragment thereof        according to embodiment 71, wherein the amino acid motif has the        sequence:

-Y-X-S(phosphorylated)-P-

-   -   -   wherein Y is tyrosine, X is a naturally occurring amino            acid, P is proline and S(phosphorylated) is serine with a            phosphorylated hydroxyl side chain.

    -   73. The monoclonal antibody or epitope-binding fragment thereof        according to any one of the preceding embodiments comprising an        Fc region.

    -   74. The monoclonal antibody or epitope-binding fragment thereof        according to any one of the preceding embodiments further        comprising a moiety for increasing the in vivo half-life of the        agent.

    -   75. The monoclonal antibody or an epitope-binding fragment        thereof according to any one of the preceding embodiments, that        specifically binds to human tau comprising a phosphorylated        residue 396 according to the test criteria: i) the antibody does        not substantially bind to non-phosphorylated tau; ii) the        antibody does not substantially bind to tau phosphorylated at        404 when 396 is not phosphorylated; iii) the antibody does bind        to tau phosphorylated at 396; and iv) the antibody does bind to        tau when both 396 and 404 are phosphorylated.

    -   76. The monoclonal antibody or an epitope-binding fragment        thereof according to any one of the preceding embodiments,        elicited against the bi-phosphorylated peptide comprising at        least 18 consecutive amino acid residues, such as at least 20        consecutive amino acid residues within        TDHGAEIVYK^({p})SPVVSGDT^({p})SPRHL (SEQ ID NO:2) covering        residues 386-408 of 2N4R tau.

    -   77. The monoclonal antibody or an epitope-binding fragment        thereof according to embodiment 76, elicited against the        bi-phosphorylated peptide comprising 18-40, such as at 18-30,        such as 20-30 amino consecutive acid residues comprising        TDHGAEIVYK^({p})SPVVSGDT^({p})SPRHL (SEQ ID NO:2) covering        residues 386-410 of 2N4R tau.

    -   78. The monoclonal antibody or an epitope-binding fragment        thereof according to any of embodiments 1 to 70, having a        specificity for phosphoTau (pTau) from AD-diseased patients over        age-matched healthy controls, such that said monoclonal antibody        or an epitope-binding fragment thereof has a specificity        difference for phosphoTau (pTau) from AD-diseased patients over        tau from age-healthy matched controls of more than 50-fold, such        as more than 100-fold increase in specificity for AD disease        material compared to healthy control material in an ELISA based        assay detect phosphoTau (pTau) in brain homogenates from AD and        from healthy control subjects, using a phospho- and        multimer-specific Setup 1 ELISA.

    -   79. The monoclonal antibody or an epitope-binding fragment        thereof according to embodiment 78, having a specificity for        AD-diseased Tau such that said monoclonal antibody or an        epitope-binding fragment thereof has a specificity difference        for AD over age-healthy matched controls of more than 50-fold,        such as more than 100-fold increase in specificity for AD        disease material compared to healthy control material in an        ELISA based assay detect phosphoTau (pTau) in brain homogenates        from AD and from healthy control subjects, using a phospho- and        multimer-specific Setup 1 ELISA.

    -   80. The monoclonal antibody or an epitope-binding fragment        thereof according to any of embodiments 1 to 70, elicited        against the bi-phosphorylated peptide:        TDHGAEIVYK^({P})SPVVSGDT^({p})SPRHL (SEQ ID NO:2) covering        residues 386-410 of 2N4R tau, or an epitope-binding fragment        thereof, capable of specifically binding to the phosphorylated        residue 396 of human tau.

    -   81. An antibody, or epitope-binding fragment thereof, as defined        according to any of embodiments 1 to 70, which has been produced        or manufactured in a cell line such as a human cell line, a        mammal non-human cell line, an insect, yeast or bacterial cell        line, or by a recombinant technology.

    -   82. The antibody, or epitope-binding fragment thereof, according        to embodiment 81 produced in a CHO cell line, HEK cell line,        BHK-21 cell line, murine cell line (such as a myeloma cell        line), fibrosarcoma cell line, PER.C6 cell line, HKB-11 cell        line, CAP cell line and HuH-7 human cell line.

    -   83. The monoclonal antibody or an epitope-binding fragment        thereof according to any of embodiments 1 to 70, wherein said        monoclonal antibody is expressed by a hybridoma that was        isolated by screening hybridomas with human pathological and        non-pathological tau to isolate clones that both i) were        specific towards the phospho-epitopes S396 and ii) specifically        recognize hyper-phosphorylated tau from human Alzheimer's        disease brains, wherein said antibodies or epitope binding        fragments thereof are able to discriminate between pathological        and non-pathological human tau protein.

    -   84. The monoclonal antibody or epitope-binding fragment thereof        according to any of embodiments 1 to 70, wherein the antibody or        epitope-binding fragment thereof further comprises a detectable        moiety.

    -   85. The monoclonal antibody or epitope-binding fragment thereof        according to embodiment 84 wherein the detectable moiety is a        fluorescent label, a chemiluminescent label, a paramagnetic        label, a radioisotopic label or an enzyme label.

    -   86. A preparation comprising the antibody or epitope-binding        fragment thereof define in any of embodiments 1 to 70, wherein        said preparation is substantially free of naturally-arising        antibodies that are either not capable of binding to tau or that        do not materially alter an anti-tau functionality of the        preparation, wherein said functionality is selected from the        group consisting of:        -   (i) a substantial inability to bind to non-phosphorylated            tau;        -   (ii) a substantial inability to bind to tau that is            phosphorylated at S404 and not phosphorylated at S396;        -   (iii) the ability to bind to tau phosphorylated at S396;        -   (iv) the ability to bind tau phosphorylated at both S396 and            at S404;        -   (v) the ability to selectively discriminate between            phosphorylated tau residues S396 and S404 such that it is            substantially unable to bind the phosphorylated 404 residue            or such that it preferentially binds to S396;        -   (vi) the ability to bind hyper-phosphorylated tau from human            Alzheimer's disease brains;        -   (vii) the ability to discriminate between pathological and            non-pathological human tau protein; and/or        -   (viii) the capability, when used as described in the            Examples with immune-depleted rTg4510 extracts from            transgenic mice, to specifically reduce the            hyperphosphorylated tau 64 kDa and 70 kDa bands by at least            90%, while not reducing the 55 kDa tau band by more than            10%.

    -   87. A preparation comprising the antibody or epitope-binding        fragment thereof defined by embodiments 1 to 70, wherein said        antibody or said epitope-binding fragment thereof possesses a        structural change in its amino acid sequence, relative to the        structure of a naturally-occurring anti-tau antibody, wherein        said structural change causes said antibody or said fragment to        exhibit an altered functionality relative to the functionality        exhibited by said naturally-occurring anti-tau antibody, wherein        said functionality is selected from the group consisting of:        -   (i) a substantial inability to bind to non-phosphorylated            tau;        -   (ii) a substantial inability to bind to tau that is            phosphorylated at S404 and not phosphorylated at S396;        -   (iii) the ability to bind to tau phosphorylated at S396;        -   (iv) the ability to bind to tau phosphorylated at both S396            and at S404;        -   (v) the ability to selectively discriminate between            phosphorylated tau residues S396 and S404 such that it is            substantially unable to bind the phosphorylated 404 residue            or such that it preferentially binds to S396;        -   (vi) the ability to bind hyper-phosphorylated tau from human            Alzheimer's disease brains;        -   (vii) the ability to discriminate between pathological and            non-pathological human tau protein; and/or        -   (viii) the capability, when used as described herein with            immune-depleted rTg4510 extracts from transgenic mice, to            specifically reduce the hyperphosphorylated tau 64 kDa and            70 kDa bands by at least 90%, while not reducing the 55 kDa            tau band by more than 10%.

    -   88. A pharmaceutical composition comprising the monoclonal        antibody or epitope-binding fragment thereof defined in any of        embodiments 1 to 70, or the preparation according to any of        embodiments 86 to 87, and a pharmaceutical acceptable carrier.

    -   89. A nucleic acid encoding a monoclonal antibody or        epitope-binding fragment thereof according to any one of        embodiments 1 to 70, or encoding a constituent chain thereof.

    -   90. The monoclonal antibody, or epitope-binding fragment        thereof, defined in any one of embodiments 1 to 70, or the        preparation according to any one of embodiments 86 to 87, or the        pharmaceutical composition of embodiment 88, for use in therapy.

    -   91. The monoclonal antibody, or epitope-binding fragment        thereof, according to any one of embodiments 1 to 70, or the        preparation according to any one of embodiments 86 to 87, or the        pharmaceutical composition of embodiment 88, for use in        treating, diagnosing or imaging a tauopathy.

    -   92. The monoclonal antibody, or epitope-binding fragment        thereof, according to any one of embodiments 1 to 70, or the        preparation according to any one of embodiments 86 to 87, or the        pharmaceutical composition of embodiment 88, for use in treating        a tauopathy selected from the group consisting of Alzheimer's        disease, Argyrophilic Grain Disease (AGD), Psychosis,        particularly Psychosis due to AD or Psychosis in patients with        AD, apathy due to AD or apathy in patients with AD, psychiatric        symptoms of patients with Lewy body dementia, Progressive        Supranuclear Palsy (PSP), Frontotemporal dementia (FTD or        variants thereof), TBI (traumatic brain injury, acute or        chronic), Corticobasal Degeneration (CBD), Picks Disease,        Primary age-related tauopathy (PART), Neurofibrillary        tangle-predominant senile dementia, Dementia pugilistica,        Chronic traumatic encephalopathy, stroke, stroke recovery,        neurodegeneration in relation to Parkinson's disease,        Parkinsonism linked to chromosome, Lytico-Bodig disease        (Parkinson-dementia complex of Guam), Ganglioglioma and        gangliocytoma, Meningioangiomatosis, Postencephalitic        parkinsonism, Subacute sclerosing panencephalitis, Huntington's        disease, lead encephalopathy, tuberous sclerosis,        Hallervorden-Spatz disease and lipofuscinosis.

    -   93. The monoclonal antibody, or epitope-binding fragment        thereof, according to any one of embodiments 1 to 70, for use in        the treatment of Alzheimer's disease.

    -   94. The monoclonal antibody, or epitope-binding fragment        thereof, according to any one of embodiments 1 to 70, or the        preparation according to any one of embodiments 86 to 87, or the        pharmaceutical composition of embodiment 88, for use in the        manufacturing of a medicament for treating, diagnosing or        imaging tauopathies.

    -   95. The monoclonal antibody, or epitope-binding fragment        thereof, according to any one of embodiments 1 to 70, or the        preparation according to any one of embodiments 86 to 87, or the        pharmaceutical composition of embodiment 88, for use as a        medicament for treating a disease selected from the group        consisting of Alzheimer's disease, Argyrophilic Grain Disease        (AGD), Psychosis, particularly Psychosis due to AD or Psychosis        in patients with AD, apathy due to AD or apathy in patients with        AD, psychiatric symptoms of patients with Lewy body dementia,        Progressive Supranuclear Palsy (PSP), Frontotemporal dementia        (FTD or variants thereof), TBI (traumatic brain injury, acute or        chronic), Corticobasal Degeneration (CBD), Picks Disease,        Primary age-related tauopathy (PART), Neurofibrillary        tangle-predominant senile dementia, Dementia pugilistica,        Chronic traumatic encephalopathy, stroke, stroke recovery,        neurodegeneration in relation to Parkinson's disease,        Parkinsonism linked to chromosome, Lytico-Bodig disease        (Parkinson-dementia complex of Guam), Ganglioglioma and        gangliocytoma, Meningioangiomatosis, Postencephalitic        parkinsonism, Subacute sclerosing panencephalitis, Huntington's        disease, lead encephalopathy, tuberous sclerosis,        Hallervorden-Spatz disease and lipofuscinosis.

    -   96. A method of treating, diagnosing or imaging Alzheimer's        disease or other tauopathies in a subject, said method        comprising administering a therapeutically effective amount of        the monoclonal antibody, or epitope-binding fragment thereof,        according to any one of embodiments 1 to 70, the preparation        according to any one of embodiments 86 to 87, or the        pharmaceutical composition of embodiment 88 to said subject.

    -   97. The method according to embodiment 96 wherein the treatment        is chronic.

    -   98. The method according to embodiment 97, wherein the chronic        treatment is for at least 2 weeks, such as at least for 1 month,        at least for 6 months, or at least for 1 year.

    -   99. The method according to any one of embodiments 96 to 98        wherein the subject is human.

    -   100. A kit comprising the antibody, or fragment thereof,        according to any one of embodiments 1-70, the preparation        according to any one of embodiments 86 to 87, or the        pharmaceutical composition of embodiment 88 for use in therapy.

    -   101. The monoclonal antibody, or epitope-binding fragment        thereof, of embodiments 1 to 70, or a preparation or        pharmaceutical composition comprising said antibody or fragment,        for use in detecting or measuring the presence or amount of said        tau in the brain of a subject.

    -   102. The monoclonal antibody, or epitope-binding fragment        thereof, preparation or pharmaceutical composition of embodiment        88, wherein said detection or measurement comprises in vivo        imaging of said anti-tau antibody bound to said tau.

    -   103. The monoclonal antibody, or epitope-binding fragment        thereof, preparation or pharmaceutical composition of embodiment        99 to 100, wherein said detection or measurement comprises ex        vivo imaging of said anti-tau antibody or said fragment thereof,        bound to said tau.

    -   104. A method of removing at least 90% of hyperphosphorylated        Tau from a tangle said tangle comprising hyperphosphorylated Tau        said method comprising contacting hyperphosphorylated Tau with a        monoclonal antibody, or epitope-binding fragment thereof, said        antibody or epitope-binding fragment thereof, being selective        for Tau having residue 396 phosphorylated and as defined in any        one of embodiments 1 to 70.

    -   105. A method of delaying the progression of Alzheimer's Disease        in a patient, said method comprising reducing or attenuating the        accumulation of pathological tau protein in said patient by        administering a monoclonal antibody, or epitope-binding fragment        thereof, said antibody or epitope-binding fragment thereof,        being selective for Tau having residue 396 phosphorylated and as        defined in any one of embodiments 1 to 70.

    -   106. A pharmaceutical composition comprising i) a Tau antibody        according to embodiment 1-70 and in particular embodiments 17,        18, 44 or 45 and ii) a compound selected from the group        consisting of        -   a) a BACE inhibitor;        -   b) a compound useful in active or passive Tau immunotherapy;        -   c) a compound useful in active or passive AR peptide            immunotherapy;        -   d) an NMDA receptor antagonists;        -   e) a further a Tau protein aggregation inhibitor;        -   e) an acetylcholine esterase inhibitor;        -   f) an antiepileptic;        -   g) an anti-inflammatory drug; and        -   h) an SSRI; and        -   iii) one or more pharmaceutically acceptable exicipients.

    -   107. A method of treating Alzheimer's Disease, reducing of the        progression of AD or reducing the symptoms of AD, comprising a        therapy comprising the administration of        -   i) a Tau antibody according to embodiment 1-70 and in            particular embodiments 17, 18, 44 or 45 and ii) a compound            selected from the group consisting of        -   a) a BACE inhibitor;        -   b) a compound useful in active or passive Tau immunotherapy;        -   c) a compound useful in active or passive AR peptide            immunotherapy;        -   d) an NMDA receptor antagonists;        -   e) a further a Tau protein aggregation inhibitor;        -   e) an acetylcholine esterase inhibitor;        -   f) an antiepileptic;        -   g) an anti-inflammatory drug; and        -   h) an SSRI.

    -   108. A kit comprising i) a composition comprising a Tau antibody        according to embodiment 1-70 and in particular embodiments 17,        18, 44 or 45 and ii) a composition a compound selected from the        group consisting of        -   a) a BACE inhibitor;        -   b) a compound useful in active or passive Tau immunotherapy;        -   c) a compound useful in active or passive AR peptide            immunotherapy;        -   d) an NMDA receptor antagonists;        -   e) a further Tau protein aggregation inhibitor;        -   f) an acetylcholine esterase inhibitor;        -   g) an antiepileptic;        -   h) an anti-inflammatory drug; and        -   i) an antidepressant.

    -   109. A composition according to claim 106, a method according to        claim 107, a kit according to claim 108 wherein said BACE1        inhibitor is a small molecule BACE I inhibitor selected from the        group consisting of LY2886721, MK-8931, AZD3293, or E2609.

    -   110. A composition according to claim 106, a method according to        claim 107, a kit according to claim 108 wherein said BACE1        inhibitor is of the formula I

-   -   -   wherein Ar is selected from the group consisting of phenyl,            pyridyl, pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl,            thiazolyl, oxazolyl, isoxazolyl, and wherein Ar is            optionally substituted with one or more substituents            selected from halogen, CN, C1-C6 alkyl, C2-C6 alkenyl, C2-C6            alkynyl, C1-C6 fluoroalkyl or C1-C6 alkoxy; and R1 is one or            more hydrogen, halogen, C1-C3 fluoroalkyl or C1-C3 alkyl;            and R2 presents hydrogen or fluoro.

    -   111. A composition, method according or a kit according to claim        110, wherein the compound of Formula I is selected from the        group consisting of

-   -   112. A composition according to claim 106, a method according to        claim 107, a kit according to claim 108 wherein said BACE1        inhibitor is of the Formula II

-   -   -   wherein Ar is selected from the group consisting of phenyl,            pyridyl, pyrimidyl, pyrazinyl, imidazolyl, pyrazolyl,            1,2,4-triazolyl, thiophenyl, thiazolyl, oxazolyl,            isoxazolyl, 1,3,4-thiadiazolyl, isothiazolyl,            1,3,4-oxadiazolyl, 1,2,4-oxadiazolyl, furazanyl and            1,2,4-thiadiazolyl and where the Ar is optionally            substituted with one or more halogen, CN, C1-C6 alkyl, C2-C6            alkenyl, C2-C6 alkynyl, C1-C6 fluoroalkyl or C1-C6 alkoxy;            R1 is C1-C3 alkyl or C1-C3 fluoroalkyl; R2 is hydrogen,            halogen, C1-C3 fluoroalkyl or C1-C3 alkyl; and R3 is C1-C3            alkyl.

    -   113. A composition according to claim 106, a method according to        claim 107, a kit according to claim 108 wherein said BACE1        inhibitor selected from the group consisting of:        N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4        fluorophenyl)-5-fluoropicolinamide;        N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-methoxypyrazine-2-carboxamide;        N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4-fluorophenyl)-5-methoxypicolinamide;        N-(3-((2R,5S)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4fluorophenyl)-5-cyano-3-methylpicolinamide;        and        N-(3-((2R,5R)-6-amino-3,3,5-trifluoro-2,5-dimethyl-2,3,4,5-tetrahydropyridin-2-yl)-4        fluorophenyl)-5-(difluoromethyl)pyrazine-2-carboxamide.

    -   114. A composition according to claim 106, a method according to        claim 107, a kit according to claim 108 wherein said NMDA        receptor antagonist is selected from the group consisting of,        Memantine, Namenda, Namzaric (memantine/donepezil), and generic        forms thereof.

    -   115. A composition according to claim 106, a method according to        claim 107, a kit according to claim 108 wherein said        acetylcholine esterase inhibitor is selected from the group        consisting of Donepezil, Galantamine and Rivastigmine.

    -   116. A composition according to claim 106, a method according to        claim 107, a kit according to claim 108 wherein said        antidepressant is selected from the group consisting of        Escitalopram, Sertraline, Citalopram, Paroxetine, Fluoxetine,        Venlafaxine, Trazodone, Mirtazapine, Vortioxetine and generic        forms thereof.

    -   117. A composition according to claim 106, a method according to        claim 107, a kit according to claim 108 for use in treating a        tauopathy selected from the group consisting of Alzheimer's        disease, Argyrophilic Grain Disease (AGD), Psychosis,        particularly Psychosis due to AD or Psychosis in patients with        AD, psychiatric symptoms of patients with Lewy body dementia,        Progressive Supranuclear Palsy (PSP), Frontotemporal dementia        (FTD or variants thereof), TBI (traumatic brain injury, acute or        chronic), Corticobasal Degeneration (CBD), Picks Disease,        Primary age-related tauopathy (PART), Neurofibrillary        tangle-predominant senile dementia, Dementia pugilistica,        Chronic traumatic encephalopathy, stroke, stroke recovery,        neurodegeneration in relation to Parkinson's disease,        Parkinsonism linked to chromosome, Lytico-Bodig disease        (Parkinson-dementia complex of Guam), Ganglioglioma and        gangliocytoma, Meningioangiomatosis, Postencephalitic        parkinsonism, Subacute sclerosing panencephalitis, Huntington's        disease, lead encephalopathy, tuberous sclerosis,        Hallervorden-Spatz disease and lipofuscinosis.

    -   118. A composition according to claim 106, a method according to        claim 107, a kit according to claim 108 for use in the        manufacturing of a medicament for treating, diagnosing or        imaging tauopathies.

    -   119. A composition according to claim 106, a method according to        claim 107, a kit according to claim 108 for treating Alzheimer's        disease, Argyrophilic Grain Disease (AGD), Progressive        Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD),        Psychosis due to AD or Psychosis in patients with AD, and        psychiatric symptoms of patients with Lewy body dementia.

EXAMPLES Example 1: Immunization of Mice with Tau Phospho-Peptides396/404

C56/BL6 and FVB mice were immunised with 10 μg P30 conjugatedphosphorylated tau peptide 386-408 (pS396/pS404) (SEQ ID NO:2)formulated in TiterMax adjuvant.

Mice (C56/BL6 and FVB strains, female and male. 2- to 3-month-old micewere immunized with peptide epitope P30 conjugated phosphorylated tau386-408.

Immunogenic P30 conjugated phosphorylated tau 386-408 (pS396/pS404)peptide was formulated in TiterMax (400 μg/ml peptide mixed 1:1 vol:vol)following the TiterMax/vendor protocol and mice were injectedsubcutaneously with 20 μg peptide (100 μl) of antigen. Control mice wereinjected with adjuvant only. All peptide-immunised mice were boostedwith 0.5 μg peptide/Titermax (10 μg/ml peptide formulated as describedabove and injected) at monthly intervals. The mice were finally boostedwith P30 conjugated phosphorylated tau 386-408 (pS396/pS404) withoutTitermax 3 days prior to harvest of splenocyte followed by fusion ofsplenocytes with SP-2 cells. Generated primary hybridomas were selectedfor re-cloning cycles after exhibiting positive binding tophosphorylated tau 386-408 (pS396/pS404) as detected by ELISA, andexhibiting preferential binding activity to S1 and P3 antigens from ADand TG4510 brain lysate (described below in Example 2B). Such bindingwas compared with the binding activity of such antibodies to brainlysate from controls, using dot blots and brain lysate coated ELISA orMSD plates.

Example 2A: Hybridoma Generation

The mice were boosted with P30 conjugated phosphorylated tau 386-408(pS396/pS404) without Titermax 3 days prior to harvest from spleens fromresponding mice followed by fusion of splenocytes with SP-2 cells.Hybridomas were selected for re-cloning cycles after positive binding tophosphorylated tau 386-408 (pS396/pS404) detected by ELISA, andpreferential binding activity to S1 and P3 antigens from AD and TG4510brain lysate in comparison to brain lysate from controls using dot blotsand brain lysate coated ELISA or MSD plates.

Example 2B: Western Blot and Dot-Blot Analysis of Specific AntibodiesTau Biochemical Fractionation

Brain tissues from humans or rTg4510 mice overexpressing the human taumutation P301L were homogenized in 10 volumes of Tris-buffered salinecontaining protease and phosphatase inhibitors as follows: 50 mMTris/HCl (pH 7.4); 274 mM NaCl; 5 mM KCl; 1% protease inhibitor mixture(Roche); 1% phosphatase inhibitor cocktail I & II (Sigma); and 1 mMphenylmethylsulfonyl fluoride (PMSF; Sigma). The homogenates werecentrifuged at 27,000×g for 20 min at 4° C. to obtain supernatant (51)and pellet fractions. Pellets were re-homogenized in 5 volumes of highsalt/sucrose buffer (0.8 M NaCl, 10% sucrose, 10 mM Tris/HCl, [pH 7.4],1 mM EGTA, 1 mM PMSF) and centrifuged as above. The supernatants werecollected and incubated with sarkosyl (1% final concentration; Sigma)for one hour at 37° C., followed by centrifugation at 150,000×g for onehour at 4° C. to obtain sarkosyl-insoluble pellets, referred to as P3fraction herein. The P3 pellet was resuspended in TE buffer (10 mMTris/HCl [pH 8.0], 1 mM EDTA) to a volume equivalent to half of theoriginal volume used for the brain homogenates.

Western and Dot Blots

Fractionated tissue extracts 51 and P3 were dissolved in SDS-samplebuffer containing 0.1 M DTT. The heat-treated samples (95° C. for 10min) were separated by gel electrophoresis on 4-12% Bis-Tris SDS-PAGEgels (Invitrogen) and transferred onto PVDF membranes (BioRadLaboratories, Hercules, Calif.). Dot blot samples were spotted directlyonto nitrocellulose membranes (Amersham, Pittsburgh, Pa.) at knownconcentrations across samples. Both Western and dot blot membranes wereblocked in 5% non-fat dry milk in TBS-Tween (0.5%) pH 7.4, followed byincubation in 1 μg/ml of C10-2 overnight at 4° C. Membranes were washedand incubated with peroxidase-conjugated anti-mouse IgG (1:5000; JacksonImmunoResearch, West Grove, Pa.). Bound antibodies were detected usingan enhanced chemiluminescence system (ECL PLUS kit; PerkinElmer).Quantitation and visual analysis of Western and dot blotimmunoreactivity was performed with a computer-linked LAS-4000Biolmaging Analyzer System (Fujifilm, Tokyo, Japan) and Multi Gauge v3.1software (Fujifilm). Protein loading was adjusted by the volume oforiginal fractions and can be converted to original tissue wet weight.

Example 3. Fluid Phase Inhibition Assay for AD-P3 Capture Example 3APurpose

To quantify inhibition of human 010-2 and mutated variants of binding topS396 tau antigens in AD-P3 brain material to murine C10.2. MSD platescoated with mouse 010-2 prior to incubation with AD-P3 or AD-P3preincubated with C10.2 variants of interest. The degree of inhibitionis depicted as 1050 values reflecting apparent affinity of fluid phaseantibody binding to antigens. 1050 values were obtained by fitting toone or two-site binding model using Graph Pad Prism software. A negativecontrol antibody (mouse 010-1) reactive with P (404) Tau was added forcomparison.

Method: MSD plates were coated with capture antibody (750 ng/ml murine010-2 in carbonated buffer pH 8.5) overnight at room temperaturefollowed by blocking (30 min. in PBS, 3% BSA, 0.1% NP40) and 5 timeswash (PBS, 0.1% BSA, 0.1% NP40). Graded concentration (0-1000 nM) ofantibodies was incubated 60 min. with AD-P3 material at room temperatureand subsequently incubated 1 hour at room temperature in MSD platescoated with mouse 010-2 as described above. Plates were washed 5 timesin (PBS, 0.1% BSA, 0-0.1% NP40) and anti-human total Tau (MSD sulfotagged 1:50) was added to detect captured tau which reflectnon-inhibited free tau antigens.

Results: Data showed dose dependent inhibition of tau capture usinghuman 010-2 and variants (FIG. 1). The variants C10-2_N32S andC10-2_N32S:A101T inhibits stronger (IC50=44 and 14 nM respectively,fitted to one site binding models) whereas C10-2 showed heterogeneousinhibition reflected by best fit to two-site binding model (IC50 14nM/630 nM). The low affinity binding (IC50=630) was predominant sincehigh affinity antibody binding (IC50=14 nM) comprised less than 25% ofthe total binding. Results are shown in FIG. 1.

Example 3B Purpose

To quantify inhibition of human 010-2 and mutated variant binding topS396 tau 386-408 peptides in fluid phase inhibition assay. The degreeof inhibition is depicted as 1050 values reflecting apparent affinity ofantibody binding. 1050 values were obtained by fitting to one ortwo-site binding model using Graph Pad Prism software. A negativecontrol antibody (mouse 010-1) reactive with P (404) Tau was added forcomparison.

Method: MSD plates were coated with pS396 Tau 386-408 peptide incarbonate buffer pH 9,5, overnight at room temperature followed byblocking (30 minutes in PBS, 3% BSA, 0.1% NP40) and 5 times wash (PBS,0.1% BSA, 0.1% NP40). Graded concentration (0-1000 nM) of pS396 tau386-408 was incubated 60 min. with 1 ng/ml antibody at room temperatureand subsequently incubated 1 hour at room temperature in MSD platescoated with pS396 Tau 386-408 as described above. Plates were washed 5times in (PBS, 0.1% BSA, 0.1% NP40) and anti-human total Tau (MSD sulfotagged 1:50) was added to detect bound antibody which reflectnon-inhibited free antibody. Results are shown in FIG. 2.

Example 4. Immunohistochemical Profiling of Antibodies Tissues

Mouse: Mouse brain tissues were collected from 8 months old rTg4510mice. These transgenic mice express human mutated Tau (P301L 0N4R) undera tet-off responsive element in CamK2 positive neurons and show apronounced tau hyperphosphorylation and tangle formation from 6 monthsof age and onwards. Non-transgenic littermates served as controls. Mousebrains were fixed by immersion in 4% paraformaldehyde and embedded inparaffin. Human: Formalin-fixed paraffin-embedded human brain samples offrontal cortex were acquired from Tissue Solutions (Glasgow, UK).Tissues from 3 donors with diagnosed end stage Alzheimer's disease (AD;Braak stage V-VI) were compared to an age-matched non-demented controldonor.

Immunohistochemistry:

Four μm thick sections of mouse and human tissues were cut on amicrotome, deparaffinized and subjected to antigen retrieval bymicrowaving the sections in 10 mM Citrate buffer, pH 6, for 10 minutes.Endogenous peroxidase was blocked with 1% hydrogen peroxide followed by5% normal swine serum in PBS, 1% BSA, 0.3% Triton X-100 (PBS-BT).Sections were incubated overnight at 4 degrees C. with hC10-2,hC10-2_N32S and hC10-2_N32S_A101T antibodies diluted in PBS-BT at therange of concentrations indicated in FIG. 1. The sections were washed inPBS, 0.25% BSA, 0.1% Triton X-100, before being incubated with abiotinylated secondary swine anti-human antibody (#B1140; Sigma-Aldrich)at 1:200 for 1 hour. Following additional washing, StreptAvidin-BiotinComplex kit (Vector Laboratories, Burlingame, Calif.) was applied andfinally, immunoreactivity was visualized with 0.05% diaminobenzidine.The sections were counterstained with hematoxylin to reveal the locationof nuclei.

Results

hC10-2, hC10-2_N32S and hC10-2_N32S_A101T labelled structures consistentwith pathological tau in 3 AD brains (i.e. tangles, neuropil threads,dystrophic neurites). The intensity of immunoreactivity wasconcentration dependent. No apparent labelling of e.g. glia cells orvessels was detected. No immunoreactivity was detected in sections froma control brain. Likewise, all 3 antibodies gave rise to the expectedpattern for phosphorylated tau in both hippocampus and cortex of rTg4510brains. In brain sections from non-transgenic mice no immunoreactivitywas detected.

Example 5. Decoration of Tau Structures in rTg4510 Mice Following i.v.Injection Method

Ten months old rTg4510 mice. These transgenic mice express human mutatedTau (P301L 0N4R) under a tet-off responsive element in CamK2 positiveneurons and show a pronounced tau hyperphosphorylation and tangleformation from 6 months of age and onwards. In addition,neurodegeneration is present at 10 months of age in rTg4510 mice inregions with strong pathology. Single transgenic tTA littermates servedas controls. The mice received a single injection via the tail vein witheither hC10-2, hC10-2_N32S or hC10-2_N32S_A101T antibodies at aconcentration of 80 mg/kg. A volume of 150 μL was injected per mouse.Three days after injection, the mice were perfused for 2 min. with PBSfollowed by 10 min perfusion with 4% paraformaldehyde. The brains werecryoprotected in 30% sucrose and cut into 40 microns free floatingcryosections. The sections were incubated with 5% normal swine serum inPBS/1% BSA/0.3% Triton X-100 for 20 min, washed in PBS and finallyincubated with AlexaFluor488-conjugated secondary anti-human IgG at1:200 (#709-545-149; Jackson ImmunoResearch Laboratories, West Grove,USA). Hoechst was used for nuclear staining. The sections were washed inPBS, mounted and examined by fluorescent microscopy.

Results

I.v. injection of hC10-2, hC10-2_N32S and hC10-2_N32S_A101T resulted inin vivo binding to target structures in hippocampus and cortex in agedrTg4510 mouse brains (FIGS. 4-7). The number of positive structuresobserved varied between individual rTg4510 animals. In tTA control mice,no specific fluorescent signals were detected after injection of any ofthe three antibodies (FIG. 4-7). Serving as a negative control,injection of a control human IgG did not result in signals in rTg4510mice. The positive signals in rTg4510 brains did not readily appear asintracellular staining and may represent extracellular tau materialreleased during the process of neurodegeneration. Collectively, thesedata suggest that hC10-2, hC10-2_N32S and hC10-2_N32S_A101T antibodiesare able to penetrate into the brain parenchyma and specificallydecorate targets in rTg4510 mice in vivo.

Example 6. Characterization of Tau Immunoreactivity in Alzheimer'sDisease Brains Tissues:

Paraffin-embedded human brain samples of frontal cortex were acquiredfrom Tissue Solutions (Glasgow, UK). Tissues from donors with diagnosedend stage Alzheimer's disease (AD; Braak stage V-VI) were included.

Immunohistochemistry

Four μm thick sections of human tissues were cut on a microtome,deparaffinized and subjected to antigen retrieval by microwaving thesections in 10 mM Citrate buffer, pH 6, for 10 minutes. Sections wereincubated with 5% normal swine serum in PBS, 1% BSA, 0.3% Triton X-100(PBS-BT) followed by overnight incubation at 4° C. with hC10-2 orhC10-2_N32S_A101T antibodies diluted in PBS-BT. The sections were washedin PBS, 0.25% BSA, 0.1% Triton X-100. Immunoreactivity was visualized byAlexaFluor488-conjugated secondary anti-human IgG (1:200; #709-545-149,Jackson ImmunoResearch Laboratories, West Grove, USA). For doubleimmunofluorescence, sections were co-incubated with AT8 (1:500; #MN1020,ThermoFisher, Waltham USA) or E1 total human tau antibody, custom maderabbit antibody raised against N-terminal tau 19-33 (Crowe et al, 1991).AT8 and E1 immunoreactivities were visualized with anti-mouseAlexaFluor568 (1:400; #A10037, ThermoFisher) and anti-rabbitAlexaFluor568 (1:400; #A10042, ThermoFisher), respectively. The sectionswere analyzed by fluorescent microscopy.

Results

In AD sections double stained for N-terminal total tau and pS396 tau apopulation of tangle-bearing neurons were labelled by both E1 as well aseither hC10-2 and hC10-2_N32S_A101T antibodies (FIG. 7). A number of tautangles were only labelled by either hC10-2 and hC10-2_N32S_A101Tantibodies (FIG. 7, arrows). Extracellular tau (ghost tangles) haspreviously been shown not to be stained by N-terminal tau antibodies(e.g. Bondareff et al, 1990; Braak et al, 1994; Flores-Rodrigues et al,2015). Thus, tau species labelled by hC10-2 or hC10-2_N32S_A101Tantibodies alone likely represent extracellular ghost tangles.

Western Blots and Immunoprecipitation Experimental Procedure andExperimental Description

Transgenic rTg4510 mice were used: a human tau cDNA with the P301Lmutation (4R0N TauP301L) was placed downstream of atetracycline-operon-responder (TRE) construct. To activate thetransgene, the responder has to be co-expressed with an activatorconstruct, consisting of the tetracycline conditional gene expressionsystem (tTA). The tTA activator system was placed downstream of theCaMKIIα promoter thus restricting the expression of TRE mainly toforebrain structures. The tau transgene responder was expressed in theFVB/N (Taconic) mouse strain, and the tTA activator system wasmaintained on 129S6 (Taconic) mouse strain. Their F1 progeny carriedresponder and activator transgenes (rTg4510) along with non-transgenic(non-tg) and single-transgenic littermate mice. Only F1 mice were usedfor the experiments. All mice were bred at Taconic, Denmark andgenotyped by the analysis of tail DNA using the primer pair's5′-GATTAACAGCGCATTAGAGCTG-3′ (SEQ ID NO:60) &5′-GCATATGATCAATTCAAGGCCGATAAG-3′ (SEQ ID NO:61) for the tTA activatortransgene and 5′-TGAACCAGGATGGCTGAGCC-3′ (SEQ ID NO:62) &5′-TTGTCATCGCTTCCAGTCCCCG-3′ (SEQ ID NO:63) for the mutant tau respondertransgene. Mice were group-housed and received water and food(Brogaarden, Denmark) ad libitum as well as enrichment materials. Thelight/dark cycle was 12 h; room temperature was 21±2° C. and a relativehumidity of 55%±5%. Experiments were performed in accordance with Danishlegislation on experimental animals (license no. 2014-15-0201-00339).

Mice were euthanized by cervical dislocation in order to preserve themetabolic environment of the brain and to prevent artefacts that couldalter the biochemical profiles of tau. Mouse brains were bisectedsagittal down the midline to yield two hemispheres. The cerebral cortexand hippocampus of the right hemisphere of each animal were quicklyfrozen on dry ice and stored at −80° C. until use. Frozen human corticesfrom Alzheimer's disease (AD) patients and aged healthy control (HC)donors were purchased from Tissue Solution (Glasgow, UK). Human brainspecimens had similar postmortem processing time <6 h, and werecharacterized for amyloid and tau pathology and selected AD specimensclassified as Braak stage V-VI.

To immunoprecipitate tau protein from brain lysates a CrosslinkImmunoprecipitation kit (Thermo Fisher Pierce 26147) was used accordingto manufacturer's instructions. Briefly, the antibody was bound toProtein A/G plus agarose followed by crosslinking of the bound antibodywith DSS (disuccinimidyl suberate). Brain homogenate was prepared inTris buffer (25 mM Tris/HCl pH 7.6, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,and complete protease and phosphatase inhibitor mix) and pre-clearedovernight at 4° C. with control agarose resin. Pre-cleared lysate wasincubated with antibody-crosslinked resin overnight at 4° C. followed byantigen elution with 50 μl elution buffer (pH 2.8) and immediatelycentrifuged into collection tubes containing 5 μl 1 M Tris, pH 9.5.Immunoprecipitated tau was dissolved in SDS-sample buffer containingdithiothreitol (DTT, 100 mM), heat-treated (95° C. for 10 min) andsubjected to Western blotting as described below.

Human tau concentrations were measured in brain homogenates andpre-cleared lysates by ELISA for total human tau according to themanufacturer's instructions (Invitrogen).

Tissues were homogenized in 10 volumes of Tris-buffered saline (TBS)containing protease and phosphatase inhibitors as follows: 50 mMTris/HCl (pH 7.4); 274 mM NaCl; 5 mM KCl; 1% protease inhibitor mixture(Roche); 1% phosphatase inhibitor cocktail I & II (Sigma); and 1 mMphenylmethylsulfonyl fluoride (PMSF). The homogenates were centrifugedat 27,000×g for 20 min. at 4° C. to obtain supernatant (51) and pelletfractions. Pellets were re-homogenized in 5 volumes of high salt/sucrosebuffer (0.8 M NaCl, 10% sucrose, 10 mM Tris/HCl, [pH 7.4], 1 mM EGTA, 1mM PMSF) and centrifuged as above. The supernatants were collected andincubated with sarkosyl (1% final concentration; Sigma) for one hour at37° C., followed by centrifugation at 150,000×g for one hour at 4° C. toobtain salt and sarkosyl-extractable (S3) and sarkosyl-insoluble (P3)fractions. The P3 pellet was resuspended in TE buffer (10 mM Tris/HCl[pH 8.0], 1 mM EDTA) to a volume equivalent to half of the originalvolume used for the brain homogenates. To enrich S1 fractions forhyperphosphorylated tau species a portion of the S1 fraction wasseparated by further centrifugation at 150,000×g for 20 min. tosupernatant (S1s) and precipitate (S1p) fractions. The S1p pellet wasre-suspended in TBS buffer to a volume equivalent to one fifth of theoriginal S1 volume used. Fractionated tissue extracts S1, S1p and P3were dissolved in SDS-sample buffer containing DTT (100 mM). Theheat-treated samples (95° C. for 10 min.) were separated by gelelectrophoresis on 4-12% Bis-Tris SDS-PAGE gels (Invitrogen) andtransferred onto PVDF membranes (BioRad Laboratories, Hercules, Calif.).After blocking with a blocking solution containing 5% nonfat milk and0.1% Triton-X100 in TBS, the membranes were incubated with 1 μg/mlhC10.2, hC10-2_N32S, hC10-2_N32S_A101T or rabbit anti-pS396 tau(Invitrogen). Membranes were washed, and incubated withperoxidase-conjugated anti-human IgG or anti-rabbit antibodies (1:5000;Jackson ImmunoResearch, West Grove, Pa.). Bound antibodies were detectedusing an enhanced chemiluminescence system (ECL PLUS kit; Perkin Elmer).Quantitation and visual analysis of Western blot immunoreactivity wasperformed with a computer-linked LAS-4000 Biolmaging Analyzer System(Fujifilm, Tokyo, Japan) and Multi Gauge v3.1 software (Fujifilm). Todetect tau protein, approximately 2 μg S1 from mouse was loaded, 20 μgS1 from human brains and equal volumes of the different fractions (S1,S1p, and P3) to SDS PAGE.

Detection of Pathological Tau by Western Blot

Forebrain homogenates pooled from three 32 weeks old rTg4510 mice andnon-transgenic (non-tg) control littermates and pooled cortical specimenfrom four AD and four healthy control (HC) donors were isolated into asoluble (S1), a TBS-soluble pellet (S1p) and a sarkosyl-insolublefraction (P3). hC10.2, hC10-2_N32S, hC10-2_N32S_A101T were used at 1μg/ml on western blot and detected pathological tau from rTg4510 miceand AD. We observed detection of 55 and 64 kDa tau in S1 and 64 and 70kDa tau in P3 and S1p fractions from 32 weeks old rTg4510 mice.Additionally, three truncated tau bands <50 kDa were observed in the P3fraction. No signal was detected in S1p and P3 from non-tg controllittermates not expressing human transgene tau. A weak signal around 50kDa was detected in S1 fraction from non-tg mice, most likelyrepresenting endogenous murine tau phosphorylated at S396 residue (FIG.8A-8C). It is summarized that hC10.2, hC10-2_N32S, hC10-2_N32S_A101Tdetected pS396 tau, and both normal phosphorylated 55 kDa andhyperphosphorylated 64 kDa tau species in rTg4510 mice. The strongestsignal was observed with hC10-2_N32S_A101T.

In S1, S1p and P3 fractions from AD donors hC10.2, hC10-2_N32S,hC10-2_N32S_A101T detected the typical AD tau smear and the pathologicalfour tau band pattern (54, 64, 69 and 74 kDa tau). As expected,sarkosyl-insoluble hyperphosphorylated tau species isolated from P3fraction were most pronounced, followed by soluble hyperphosphorylatedtau species enriched in the S1p fraction. No signal was detected in P3fractions from healthy control (HC). In S1 and S1p fractions from HC aweak signal around 55 kDa was detected, likely representing normalphosphorylated tau at S396 residue (FIG. 8A-8C). It is summarized thathC10.2, hC10-2_N32S, hC10-2_N32S_A101T detected the typical tau smearcharacteristic for AD and the pathological four tau band patternsrepresenting hyperphosphorylated tau. Strongest signal was observed withhC10-2_N32S_A101T.

Immunoprecipitation of Pathological Tau

To determine the ability of hC10.2, hC10-2_N32S, hC10-2_N32S_A101T tobind tau under non-denaturing conditions, a tau immunoprecipitation (IP)protocol was established where tau antibodies are covalentlycross-linked onto protein A/G resin and thereby gives IPs free fromantibody contaminations. For tau analysis by SDS-PAGE the presence ofthe heavy chains of the antibody used for IP can obtrude signals sinceboth proteins are detected around 50 kDa. The efficacy of hC10.2,hC10-2_N32S, hC10-2_N32S_A101T to pull down pathological tau from humanbrain was investigated. As antigen, 500 μg pre-cleared lysate from brainhomogenates was used from four pooled AD and HC donors containing 0.1 μgand 0.15 μg human tau (determined by human tau ELISA), respectively.hC10.2, hC10-2_N32S, hC10-2_N32S_A101T (10 μg) pulled down 54, 64, 69and 74 kDa tau species (the four pathological tau bands) and AD smearfrom pre-cleared AD homogenates (antigen/ab 1:100 ratio) visualized by apolyclonal rabbit anti-pS396 tau antibody (FIG. 9). Comparing theintensity of the tau bands from AD brains pulled down with hC10.2,hC10-2_N32S, hC10-2_N32S_A101T to control human IgG antibody and to HCbrain, it can be summarized that hC10.2, hC10-2_N32S, hC10-2_N32S_A101Timmunoprecipitated hyperphosphorylated tau at the pS396 site exclusivelyfrom AD brains and were effective at an antigen/antibody ratio of 1:100.

Cell and Aggregation Assay

HEK293 cells were transiently transfected with human tau-P301L-FLAG in6-well plates 24 hours after plating, followed 24 hours later byincubation with brain homogenate for 24 h, followed by splitting andre-plating cells and harvesting after an additional 24 hours. Cells werelysed and sonicated in PBS, supplemented with 1% triton X, Phos-stop andcomplete phosphatase and protease inhibitors (Roche) buffer andultracentrifugated at 100,000×g for 30 min. The pellet was resuspendedin SDS, sonicated and ultracentrifugated for 30 min. at 100,000×g.Supernatants were analyzed by western blotting. Cells expressing humantau-P301L showed insoluble (SDS fraction, E1/FLAG detection),hyperphosphorylated (pS396 detection) tau upon seeding with total brainhomogenates from rTg4510 tau transgenic mice.

Cells treated with control brain homogenate from tTA mice showed anabsence of aggregated hyperphosphorylated human tau. Additionally, totalcell lysates of HEK293 cells were analyzed using the tau aggregationassay from Cisbio. This assay is based on time-resolved fluorescenceusing the same antibody for both donor (Tb3+ conjugated) and acceptor(d2 conjugated) Ab in FRET. A 10 μl sample was mixed with 10 μl antibodymix and incubated for 20 hours. The plate was read on the Pherastarplate reader to assess time-resolved fluorescence (FRET signalmeasured/integrated after switching of the excitation light). The assaymeasures aggregated tau both in human autopsy material, rTg4510 mice andin seeded HEK cells with high specificity and sensitivity. Results areshown in FIG. 10.

Example 7. Immunodepletion of Tau

Alzheimer brain extracts were made from frozen post mortem prefrontalcortex in 10× volume sterile cold PBS. The tissue was homogenized usinga knife homogenizer followed by sonication, 5×0.9 second pulses atoutput 2 (Branson sonifier). The homogenate was then centrifuged at 3000g for 5 minutes at 4 C. The supernatants were aliquoted, snap frozen andstored at −80 degrees C. until use.

25 μg antibody (humanized C10-2 variants and 2.10.3, mouse AT8, ThermoScientific mn 1020) were immobilized to 125 μl of Magnetic dynabeadsuspension (Immunoprecipitation Kit Dynabeads Protein G Novex, Cat no10007D). After thorough washing the coated beads were mixed withvariable amounts of non-coated, washed beads. Starting from 100% Abcoated beads, corresponding to 5 μg antibody, down to 100% non-coatedbeads. The total amount of beads was the same in all samples. The beadswere mixed with 20 μl AD extract and incubated at room temperature for10 min. The magnetic beads were separated from the extract and theextracts were aliquoted, snap frozen and kept at −80 degrees C. untiluse.

Analysis of Depletion Using Western Blot

Samples were boiled in 1×SDS loading buffer and 100 mM DTT. A volumecorresponding to 3 μl of extracts were loaded on a 4-12% Bis-Tris NuPAGEGel (LifeTech Novex). After electrophoresis, the proteins were blottedover to an Immobilon-FL PVDF membrane (0.45 μm, IPFL10100, Millipore).The membrane was blocked with SEA blocking buffer (Prod#37527, Thermo).Tau and P-tau levels were assessed in the samples using Tau5 (Tau5 is acommercially available anti-tau antibody whose epitope is described asbeing in the middle of Tau amino acids 210-241. It is a mouse monoclonalto Tau. Abcam ab80579, 1:2000) mouse C10-2 (1 μg/ml), P-S199/202(Invitrogen 44768 G, 1:1000), P-S422 (Abcam ab79415, 1:750), human IPN(1 μg/ml). Gapdh and actin were used as loading controls (Abcam ab9484,1:2000, Sigma A5441, 1:20000). Secondary fluorophore conjugated IgGantibodies was used (IRDye 800CW Goat anti-Human, IRDye 800CW, Goatanti-rabbit, IRDye 680 Goat anti-mouse, LI-COR biosciences) and thesignal was quantified using Odyssey CLx and Image studio software(LI-COR biosciences). Quantification of individual bands as well assignal in whole lanes was done and from this sigmoidal dose-responsecurves were plotted and when possible max effect and EC50 values wereestimated.

Results

2.10.3 and C10-2 antibodies both remove a small fraction of tau from theAlzheimer's disease brain preparation. This demonstrates selectivity fora subset of tau within the total tau protein content. 2.10.3, designedto have specificity for pS422 tau removes up to 24% of the total tauamount, while 010-2 removes up to 15% of the total tau (see FIG. 11).This may be interpreted that the pS396 subset is a smaller subset oftau, all other factors being equal. Alternatively, the data mayinterpreted that the C10-2 antibodies are more selective for pS396 thanthe 2.10.3 antibody is selective for pS422.

2.10.3 and C10-2 both remove more than 90% of the tau phosphorylated atSerine 422, although the amount of antibody required to remove 50% ofthe pS422 tau differ, for 2.10.3, 0,42 μg antibody and for C10-2, 0.27μg was needed for the same effect (see FIG. 12). In one embodiment ofthe invention, the antibody is specific for an epitope within 386-404wherein serine residue 396 of human tau is phosphorylated and wherein80% of pS422 tau is removed (in immunodepletion studies by Western blotanalysis) using less than 1 μg of the antibody.

C10-2 efficiently removes tau which is phosphorylated at serine 396 (Maxeffect: 88% and half of the effect is reached by using 0.30 μgantibody). 2.10.3 removes a smaller fraction of tau being phosphorylatedat the serine 396 (Max effect: 60% and half of that effect is reachedwhen using 0.63 μg antibody) (see FIG. 13). This indicates that all Taubeing phosphorylated at serine 422, also is phosphorylated at serine396, but that there is a portion of hyperphosphorylated tau beingphosphorylated at serine 396 where the phosphorylated serine at position422 is not present. In one embodiment of the invention, the antibody isspecific for an epitope within 386-404 wherein residue 396 of human tauis phosphorylated and wherein 80% of pS396 tau is removed (inimmunodepletion studies by Western blot analysis) using less than 1 μgof the antibody

A large portion of the tau, being removed by C10-2, is alsophosphorylated at Serine 199/202, since 69% of the tau having thatphosphorylation is affected by the immunodepletion (50% of the effectwhen using 0.34 μg antibody). The 2.10.3 immunodepletion does not give asigmoidal dose response on the pS199/202 tau, although a drop in signalis seen with increasing amount of antibody (max 52% reduction when usingthe max amount of antibody (5 μg) (see FIG. 14). In one embodiment ofthe invention, the antibody is specific for an epitope within 386-404wherein Serine residue 396 of human tau is phosphorylated and wherein80% of P-S199/202 tau is removed (in immunodepletion studies by Westernblot analysis) using less than 1 μg of the antibody.

These results indicate that the C10-2 antibody targeting thephosphorylated serine 396 binds a larger pool of the hyperphosphorylatedtau then the 2.10.3 antibody targeting the phosphorylated serine at the422 position.

In investigating individual bands on western blot after immunodepletiona 25 kDa band was identified as phosphorylated at Serine 396. Thisfragment was immunodepleted by C10-2, but 2.10.3 and AT8 did not depletethis fragment (see FIG. 15). Thus, C10-2 has a unique feature removingthis truncated form of tau from Alzheimers diseased brain extracts.

Example 8. Comparing C10-2 Variants

All C10-2 variants had the same efficiency in the immunodepletion assay(see FIG. 16). These results demonstrate that the introduced mutationshas not changed the functional binding to Alzheimer brain specific Tau.

8a. Antibody Treatment in Seeded rTg4510 Mice

Transgenic mice expressing human mutated Tau (P301L 0N4R) under atet-off responsive element in CamK2 positive neurons (rTg4510) was used.This model normally starts developing Tau pathology at 3 months of age,but by feeding the mothers with doxycycline during pregnancy and for thefirst 3 weeks of the pup's life, the pathology develops at a later stage(starting after 6 months of age). The doxycycline pre-treated mice werechronically treated with mC10-2, hC10-2, 2.10.3 or control antibody, 15mg/kg/week starting at 2 months of age. At 2.5 months, Alzheimer brainextract was infused into the hippocampus. Mice were 20 anesthetized byisoflouran inhalation fixed in a stereotactic frame. The scull wasexposed and adjusted until bregma and lambda was in level. A hole wasdrilled in the scull 2 mm lateral (right) and 2.4 mm posterior of thebregma. A 10 μl syringe beveled tip (SGE) was used to inject the seedingmaterial 1.4 mm ventral to the brain surface at the at the abovementioned co-ordinates. 2 μl of the extracts, described in Examples 7,was slowly infused at the site (1 μl/minute) and the syringe was leftfor 5 minutes before removing it. The wound was closed by stiches andmice were heated while waking up. The mice were housed for 3 months andthen sacrificed and perfusion fixed with 4%. The mice were treated withantibody until sacrifice, 3 months after seeding.

Immunohistochemistry

Fixed brains were cut into 35 μm coronal sections at NSA 30 and every6th section was stained for tau tangles (Gallyas silver stain).Positively stained neurons (soma) were counted in ipsi and contralateralsides of hippocampi of all brains. All sub-regions of hippocampus wereincluded. Eight sections were counted per brain. Results reflect the sumof positive neurons from the 8 sections.

Statistical analysis: The variance is significantly different whencomparing the groups. Thus the non-parametric, Kruskal-Wallis test andDunn's multiple comparison test was used.

Results:

The extracts caused seeding of tangle pathology in the ipsilateralhippocampus. The mC10-2 treatment significantly reduced tangle pathologyin the seeded hippocampus by 57% (P<0,05). There was a clear trendindicating hC10-2 reduced pathology. 2.10.3 failed to show an effect(see FIG. 17).

Example 8b. The Anti-Seeding Effects of the C10.2 Variants

Study Objective

Transcellular propagation of tau aggregates has been suggested tocontribute to the pathological development and topological spread ofAlzheimer's disease within the CNS. In vitro seeding models wereestablished whereby cellular human tau expressed in HEK293 cells isseeded with extracellularly applied pathological tau aggregates. Thepurpose of this study was to evaluate and compare the therapeutic effectof the monoclonal human C10.2 and variants upon seeding in a treatmentparadigm. Seeding is intended to mean the introduction of an amount ofhyperphosphorylated tau (seed) that initiateshyperphosphorylation/misfolding of cellularly expressed tau.

Background Establishing Relevance of the Study

Depositions of extracellar plaques of A13 and intra-neuronal pairedhelical filaments of tau are hallmarks of Alzheimer's disease. Theintra-neuronal inclusions of tau are primarily made up of detergentinsoluble, hyperphosphorylated, amyloid forms of tau and are in ADpatients deposited in a spacio-temporal pattern suggestive of spreadingof aggregates within the CNS. Tau aggregates have experimentally beenshown to propagate from cells to cells both in vivo and in vitro in aprion-like mechanism. This presence of extra-cellular spreadingimpacting disease propagation opens up for immunotherapy with CNSpenetrant antibodies.

Application of tau aggregates/seeds have been shown to lead toaggregation of cellular tau in vitro (Frost et al., 2009, Guo and Lee2011, Yanamandra et al., 2013). We have established an in vitro seedingmodel, whereby crude brain homogenates from Tg4510 mice (containingaggregated human tau) is used to seed human 0N4R tau with a P301Lmutation transiently expressed in HEK293 cells. Importantly, no residualtau aggregates (seeds) can be detected in seeded pcDNA control cells, soall readouts are detecting the conversion of cellular tau and do notdetect any signal from the exogenous seeding material. Anti-seedingeffects of different variants of hC10.2 were determined in this assay.

Variants

Antibody C10-2 (hC10.2)Antibody N32S (hC10.2 N32S)Antibody N32Q (hC10.2 N32Q)Antibody N32S, D55E (hC10.2 N32S D55E)Antibody N32Q, D55E (hC10.2 N32Q D55E)Antibody N32S, A101T (hC10.2 N32S A101T)

Reference item(s)

Control hIgG1 (B12)

Test system/Animals

HEK293 cells transiently transfected with hTau-P301L (0N4R).

Experimental Design

Seeding material: Tg4510 homogenates (and controls) from 12 month oldmice were homogenized as 10% homogenates in TBS without inhibitors bybead homogenization and sonicated. Homogenates were spun at 21000×g for15 min. and the soluble fraction was used for seeding.

HEK293 Seeding assay: In this assay 600,000 HEK293 cells are plated perwell in a 6-well plate on day 0. On day 1, cells are transfected with 4μg plasmid DNA using 10 μl lipofectamine2000 according to manufacturer'sprotocol. Medium is changed after 4 h. On day 2 cells are seeded withcrude brain homogenate from 12 month old Tg4510 or tTa mice. Homogenatescontaining 40 μg total protein (approximately 65 ng total human tau forthe tg4510 homogenates) are applied to the medium of each well. Forantibody treatment experiments homogenates are pre-incubated with orwithout antibodies o/n at 4 degrees. 6 h post-seeding medium is changedto low serum medium to reduce cell proliferation and the antibodies arere-applied.

On day 3 (24 h post-seeding), cells are trypsinized for 3 minutes todegrade extra-cellular seeds and re-plated at; 800,000 cells per well in6-well plates for fractionation experiments, 20,000 cells per 96-wellfor the Cisbio tau aggregation assay and at 10,000 cells per 96-well forantibody uptake evaluation by high-content imaging. Antibodies arere-applied to each well.

Cellular fractionation: 48 h post-seeding, cells for fractionation areharvested by scraping in cold PBS, pelleted and lyzed in TBS with 1%triton-X with phosphatase and protease inhibitors and sonicated. Afterultracentrifugation (100,000×g for 30 min. at 4° C.) the pellet isresuspended in 1% SDS, sonicated and ultra-centrifuged once again. TheTriton-X and SDS-soluble fractions are analysed by western blotting fortotal tau (E1) and phosphorylation of tau on S396 (D1.2). D1.2 signal isquantified by immunofluorescence on the Odyssey imagingmachine.

Cisbio tau aggregation assay: 48 h post-seeding, cells for the Cisbioaggregation assay is washed in ice-cold PBS and frozen dry. Cells arelyzed in 1% triton-X with phosphatase/protease inhibitors and benzonase(an RNase and DNase) and incubated on an orbital shaker, at 650 RPM for40 min. at 4 degrees C. Total cell lysate is analysed for tau aggregatesby the Cisbio tau aggregation assay, an assay based on using the sameantibody coupled to a donor and an acceptor for FRET measurements usinghomogenous time resolved fluorescence (HTRF®, Cisbio). Monomeric tau canonly bind either the acceptor or the donor antibody, as they arecompeting for the same binding epitope=no FRET. In contrast oligomerictau can bind both the donor and the acceptor=FRET signal. Total proteinis determined for all samples by BCA and the signal-to-noise ratio ofthe sample is normalized to protein and plotted as relative tauaggregation using 8 technical replicates.

Antibody uptake: 48 h post-seeding, plates for antibody uptake was fixedin 4% paraformaldehyde and 4% sucrose and stained with an anti-humansecondary antibody. Antibody uptake was confirmed using Cellomics.

Data analysis/Statistics

Data are presented as the pooled data of four independent biologicalreplicas performed and analysed over two different weeks +/−S.E.M. Dataare analysed using a One-way ANOVA with a Tukey's multiple comparisontest, *P<0.05, **P<0.01, ***P<0.001.

Results

hC10.2 had a reductive impact on seeding and insolublehyperphosphorylated tau by approximately 40% compared to the control.All other antibodies showed a similar or superior effect to hC10.2. Inparticular, the N32S and N32S_A101T variants of hC10.2 showed strongereffects 45% and 62% reduction in aggregated tau. The N32S_A101T variantshowed a significant stronger effect on aggregation compared to hC10.2.

Example 8C: Stability Studies on hC10.2 and Variants of the InventionStudy Objective

This study was performed to evaluate the basic stability issues with theantibodies of the invention, focusing on stressed conditions to revealany potential differences in the variants.

Study Design

All samples were initially prepared into identical starting conditions(buffer, concentration and aggregate level). This would eliminatedifferences in initial properties that potentially could influencesubsequent development of the sample behaviour. A control was kept at−80° C. and analyzed in parallel with the stressed samples (40° C.). Thesamples were removed from 40° C. at different time point and analyzedsimultaneously at the end by SEC-uplc, peptide mapping by Liquidchromatography-mass spectrometry (LCMS) and differential scanningfluorimetry (DSF).

Variants

Antibody C10-2 (hC10.2)Antibody N32S (hC10.2 N32S)Antibody N32Q (hC10.2 N32Q)Antibody N32S, D55E (hC10.2 N32S D55E)Antibody N32Q, D55E (hC10.2 N32Q D55E)Antibody N32S, A101 (hC10.2 N32S A101T)Antibody A101T (hC10.2 A101T)Antibody D55E (hC10.2 D55E)Antibody N32Q, A101T (hC10.2 N32Q A101T)

Reference Item

Antibody samples stored at −80° C. for the duration of the study wereused for reference.

Background Information

Stability aspects of a monoclonal antibody are important for bothaspects of manufacturing, final storage and formulation of drugsubstance. In this study, we have stressed the antibodies by freeze-thawcycles, high temperature and low pH in order to reveal if obviousproblems is apparent in any of the candidates.

Materials and Methods

For deamidation studies, samples are vialed in 1 ml samples andincubated at 40° C. At predefined time points a sample is moved to −80°C. and stored until analysis.

Deamidation Studies

Deamidation of Asn residues are studied at the peptide level in order toget detailed information on the actual residues in question. The mAb istherefore digested with porcine trypsin after reduction and alkylation(iodoacetic acid) using standard protocols. Peptides were separated on aCSHT130 C18 1.7 μm column and introduced to a XEVO QTOF (Waters) MSinstrument operating in MSe mode. Identical parameters were usedthroughout the series. Peptide maps from all time points were analyzedin BiopharmaLynx and quantified with the following limitations: Onlypositively identified deamidated peptides are included, no in sourcefragments are included. The % deamidation of each identified peptide ismonitored over time.

SEC Method Outline

Aggregation was determined by SEC chromatography on an ACQUITY UPLC®BEH200 SEC 1.7 μm 4.6×150 mm column in an Acquity UPLC with TUV detector(waters). Samples of 20 μl adjusted to 100 μg/ml (diluted in runningbuffer: Gibco PBS—Invitrogen #14190-094+0.1 M NaCl) were applied to thecolumn at 0.4 ml/min and separated over 6 min. by isocratic elution.

Data are analyzed by MassLynx and AUC is used to quantify levels ofaggregation.

Low pH Experiment

hC10.2 was applied to a protein G column and eluted at pH 2.8 usingstandard conditions. Sample was kept at pH 2.8 and aliquots removed attime 0, 15, 30, 60, 120 and 180 min. and neutralized before desaltedinto PBS. Samples were analyzed for binding affinity, aggregation anddeamidation.

Characterization of hC10.2 at stressed conditions: Deamidation at 40° C.and 28 days

Deamidation of Asn is observed at several minor and three major sitescovered by peptides LC:T2, HC:T36 and HC:T25. In these deamidationincrease over time, ending at 75%, 38% and 28% respectively. In contrastto peptide HC:T36 and T25, the Asn residues (LC N32 and N34) in theLC:T2 peptide was not expected, based on the in silico analysis(sequence motif), to be prone to deamidation, but surprisingly in theactual experiment they clearly differentiate from the other Asn residuesin the hC10.2. Increased deamidation of this peptide correlates with areduction in binding activity against the double phosphorylated peptideand suggests a mechanistic relationship between deamidation and IC50.This observation also suggests that changing the Asn residues 32 and/or34 in hC10.2 LC could reduce the deamidation at these sites.

In the analysis of the variants, it is clear that simply modifying theAsn32 to either Ser or Gln completely prevents the deamidation reactionon the Asn34 (FIG. 20). Also we do not detect any deamidation of Gln32in variants with this mutation.

Deamidation at Low pH

In this study, we analyzed hC10.2 in the same way as described above forthe 40° C. stability study. Only in this case a mix of trypsin and Lys-Cwas used (Promega product) to optimize cleavage at Lysine residues. Noadditional sites or degree of deamidation were observed and again themost prominent peptides were LC-T2, HC-T25 and HC-T36. In addition nochange in IC50 was observed in low pH treated samples.

SEC Analysis

Level of aggregation was below 3% in all preparations and no change indegree of aggregation was observed over 28 days at 40° C.

Example 9

Isolation of Tau Seeds from rTg4510 Mice

The rTg4510 mice are double transgenic co-expressing the 4R0N tau withP301L mutation in the human MAPT gene downstream of atetracycline-operon-responder (TRE) and an activator construct,consisting of the tetracycline conditional gene expression system (tTA).The P301L mutation is a dominant mutation leading to frontotemporaldementia with Parkinsonism linked to chromosome 17 (FTDP-17). In rTg4510mice, expression of P301L hTau induces tau pathology includingpre-tangle and neurofibrillary tangle (NFT) formation, neuronal loss andbehavioural abnormalities in an age-dependent manner. NFTs areintraneuronal tau aggregates made of paired helical filaments (PHF),twisted ribbons, or straight filaments, are detergent-insoluble andcontain predominantly hyperphosphorylated tau. Hyperphosphorylationmeans that tau is phosphorylated at more sites than tau from adulthealthy brain and that for a given site a higher than normal percentageof tau molecules is phosphorylated (>8 phospho mole/tau mole). Theanatomical distribution of NFTs is the post-mortem histopathologicalhallmark that correlates with cognitive decline in Alzheimer's disease(AD) and to memory loss in normal aging and mild cognitive impairment.The distribution pattern of NFT in the brain of AD patients is highlyhierarchical and has been divided into six stages. The gradual invasionof the brain by NFT changes has also been confirmed biochemically andhas been classified into ten stages according to the affected region.Biochemical characterization of the different tau species from AD brainswas originally based on a fractionation protocol using 1% sarkosyl forisolation of insoluble tau. Based on this method, the sarkosyl-insolublehyperphosphorylated tau species isolated in the sarkosyl-insolublepellet (P3) fraction are defined as PHF-tau and regarded as thebiochemical NFT equivalent. The buffer soluble hyperphosphorylated tauspecies isolated in the soluble fraction (S1) are defined as non-fibriloligomer tau species and regarded as the biochemical pre-tangle tauequivalent. In the rTg4510 mice, normal monomer phosphorylated andnon-phosphorylated human 4R0N transgenic tau is present in the solublefraction (S1) and visualized as 55 kDa tau species on SDS page.Hyperphosphorylated 4R0N tau with P301L mutation is displayed asmobility shifted tau of 64 kDa and 70 kDa in the soluble (S1), andexclusively in the Tris-buffered saline (TBS)-soluble precipitate (S1p)and the sarkosyl-insoluble pellet (P3) fraction. The sarkosyl-insoluble64 kDa and 70 kDa tau species isolated in P3 are tau fibrils (PHF-tau)and the biochemical NFT equivalent. The buffer soluble 64 kDa and 70 kDatau species in S1 and enriched in the TBS-soluble precipitate fraction(S1p) are oligomeric tau and the biochemical pre-tangle tau equivalent.The 70 kDa tau species are specific for the P301L mutant and also foundin FTDP-17 patients' brains. Brain tissue from 40 weeks old rTg4510 micewas homogenized in 10 volumes of TBS containing 50 mM Tris/HCl (pH 7.4),274 mM NaCl, and 5 mM KCl. The homogenates were centrifuged at 27,000×gfor 20 min at 4° C. to obtain supernatant (S1) and pellet (P1)fractions. TBS-extractable S1 fraction was separated by centrifugationat 150,000×g for one hour at 4° C. into supernatant (S1s) andprecipitate (Sip) fractions. The S1p pellet was re-suspended in 10 mMTris/HCl [pH 8.0] to a volume equivalent to a fifth of the original S1volume. The P1 pellet was re-homogenized in 5 volumes of highsalt/sucrose buffer (0.8 M NaCl, 10% sucrose, 10 mM Tris/HCl, [pH 7.4])and centrifuged at 27,000×g for 20 min at 4° C. The supernatants werecollected and incubated with sarkosyl (1% final concentration; Sigma)for one hour at 37° C., followed by centrifugation at 150,000×g for onehour at 4° C. to obtain sarkosyl-insoluble pellets, referred to as P3fraction. The P3 pellet was resuspended in 10 mM Tris/HCl [pH 8.0] to avolume equivalent to half of the original volume used for the brainhomogenates. Pathological tau from rTg4510 mouse brain characterized ashyperphosphorylated tau of 64 and 70 kDa on SDS-page was exclusivelypresent in the S1p and P3 fractions as TBS soluble oligomer andsarkosyl-insoluble fibril tau, respectively. The S1s fractions containnormal non- and phosphorylated tau displayed as 55 kDa tau on SDS-page.Pathological tau, soluble oligomeric and insoluble fibril isolated inS1p and P3 fractions respectively, was employed as hyperphosphorylatedtau seeds to recruit endogenous monomeric human tau into inclusions ofaggregated tau and to induce tau seeding in primary cortical culturesisolated from rTg4510 mice.

Seeding Assay in Primary Neuronal Culture Isolated from rTg4510 Mice

Murine cortical neurons (CTX) were isolated from day E14-16 rTg4510mouse embryos. Single transgenic tTA activator mice were time-mated withsingle transgenic mutant tau responder mice. Pregnant females wereeuthanized at 14-16 days post conception and embryos were genotyped withbrain DNA using the primer pair's 5′-GATTAACAGCGCATTAGAGCTG-3′ (SEQ IDNO:60) and 5′-GCATATGATCAATTCAAGGCCGATAAG-3′ (SEQ ID N0:61) for the tTAactivator transgene and 5′-TGAACCAGGATGGCTGAGCC-3′ (SEQ ID NO:62) and5′-TTGTCATCGCTTCCAGTCCCCG-3′ (SEQ ID NO:63) for the mutant tau respondertransgene while isolated embryonic cortices were kept in Hibernate Ewithout calcium chloride (BrainBits LLC) at 4° C. Cortices from rTg4510mouse embryos were selected and dissociated neurons plated on 100 μg/mlpoly-L-lysine coated dishes at a density of 0.13×106 cells/cm2 (420,000cells/ml, 100 μl/well, 96-well plate) and cultured in glia conditionedNeurobasal medium supplemented with 2% B-27 supplement withantioxidants, 0.5 mM L-glutamine, 100 U/ml penicillin, 0.1 mg/mlstreptomycin (all solutions from Gibco-BRL Invitrogen). Glia conditionedNeurobasal medium was generated by confluent and non-proliferatingprimary murine astrocyte cultures after 24 h incubation. Neurons werefed at 4 days in vitro (DIV) by replacing half of the medium with freshglia conditioned Neurobasal medium and thereafter feeding was performedevery seventh day. At DIV4 after feeding neuronal culture was treatedwith 1 μM cytosine arabinoside to halt proliferating cells. Theproportion of glia cells in the cultures was less than 10%, as assessedby an antibody against glia-fibrillary-acidic protein (GFAP) at DIV15.CTX from rTg4510 mice express endogenous murine tau and transgenic humantau 4R0N. CTX from rTg4510 mice contain only normal human monomeric non-and phosphorylated tau displayed as 55 kDa tau bands on SDS-page; nohyperphosphorylated tau species (64 and 70 kDa) are present in naïve CTXfrom rTg4510 mice. At DIV7 tau seeding was induced by incubating CTXwith pathological tau seeds, either soluble oligomeric or insolublefibril hyperphosphorylated tau isolated in S1p and P3 fractionsrespectively. A complete medium change was introduced at DIV11 toprevent continuous uptake of pathological tau seeds and tau seeding inthe CTX was measured at DIV15. Tau seeding was characterized byrecruitment of monomeric soluble human tau into inclusions of aggregatedand hyperphosphorylated tau displayed as mobility shifted tau bands athigher molecular weight of 64, 70 and 140 kDa on SDS page. Bothhyperphosphorylated tau seeds, soluble oligomeric or insoluble fibrilfrom S1p and P3 fractions, respectively, induced tau seeding equally inthe CTX. To investigate the effect of tau antibodies on tau seeding, CTXwere treated with a mixture of 0.1 μl P3 or 0.2 μl S1p fractions(containing 0.2 ng total human tau) isolated from 40 weeks old rTg4510mice and 10 μg antibody (hC10.2, hC10.2_N32S, hC10.2_A101T_N32S, humanIgG control) or phosphate-buffered saline (PBS). The tau seed-antibodymixture was pre-incubated for 2 h at 4° C. before addition to CTX. AtDIV11 a complete medium change to fresh glia conditioned Neurobasalmedium was performed. At DIV15 neurons were lysed in ice-cold tritonlysis buffer (1% triton X-100 in 50 mM Tris, 150 mM NaCl (pH 7.6) with1% protease inhibitor mixture (Roche), 1% phosphatase inhibitor cocktailI & II (Sigma), and 0.2% Benzonase (Sigma) under shaking at 200 rpm for45 min at 4° C. and lysates were used in the Cisbio in vitro aggregationassay and the protein content was determined by bicinchoninic acid (BCA)assay according to manufactures' instructions. The tau aggregation assayfrom Cisbio is based on time-resolved fluorescence using the sameantibody for both the donor (Tb3+ conjugated) and acceptor (d2conjugated) antibody in fluorescence resonance energy transfer (FRET)and performed according to manufactures' instructions. Briefly, 9 μlsample was mixed with 9 μl antibody mix and incubated for 20 h. Theplate was read on a Pherastar plate reader to assess time-resolvedfluorescence (FRET signal measured/integrated after switching of theexcitation light). The assay measures tau aggregation, both tauoligomers and fibrils, in brain material from rTg4510 mice and inneuronal lysates from tau seeded CTX isolated from rTg4510 embryos witha high specificity and sensitivity. Results can be seen in FIGS.21A-21B. Incubation of P3 or S1p seeds with human IgG control antibodyled to similar tau aggregation signal from seeded CTX as incubation ofP3 or S1p seeds with PBS. Signals from PBS and human IgG controlantibody were averaged and set as 100% tau aggregation. The tauantibodies hC10.2, hC10.2N32S and hC10.2A101T-N32S reduced significantlytau aggregation signals both from P3 and S1p induced tau seeding atDIV15. Results from 2 individual experiments were summarized. The hC10.2antibody reduced P3 and S1p induced tau seeding by 23%. VariantshC10.2N32S and hC10.2A101T-N32S reduced P3 and S1p induced tau seedingby 41-53% and 48-60%, respectively. Variants hC10.2N32S andhC10.2A101T-N32S were superior compared to hC10.2 in decreasing tauseeding in CTX from rTg4510 induced by hyperphosphorylated tau seedsfrom S1p or P3 consisting of oligomer or fibril tau, respectively.

Example 10

Dose-dependent decoration of tau structures in rTg4510 mice followingi.v. injection of hC10-2_N32S_A101T

Method:

Twelve months old rTg4510 mice. These transgenic mice express humanmutated Tau (P301L 0N4R) under a tet-off responsive element in CamK2positive neurons and show a pronounced tau hyperphosphorylation andtangle formation from 6 months of age and onwards. In addition,neurodegeneration is present at 12 months of age in rTg4510 mice inregions with strong pathology. The mice received a single i.v. injectionvia the tail vein with hC10-2_N32S antibodies at doses 80 mg/kg, 20mg/kg, 8 mg/kg and 0.8 mg/kg. A volume of 100 microL was injected permouse. Three days after injection, the mice were perfused for 2 min withPBS followed by 10 min perfusion with 4% paraformaldehyde. The brainswere cryoprotected in 30% sucrose and cut into 40 microns free floatingcryosections. The sections were incubated with 5% normal swine serum inPBS/1% BSA/0.3% Triton X-100 for 20 min, washed in PBS and finallyincubated with AlexaFluor488-conjugated secondary anti-human IgG at1:200 (#709-545-149; Jackson ImmunoResearch Laboratories, West Grove,USA). The sections were washed in PBS, mounted and examined byfluorescent microscopy.

Results:

I.v. injection of hC10-2_N32S resulted in in vivo binding to targetstructures mainly in hippocampus and cortex in aged rTg4510 mouse brains(FIG. 1). The number of positive structures observed varied betweenindividual rTg4510 animals. The positive signals in rTg4510 brains didnot readily appear as intracellular staining and may representextracellular tau material released during the process ofneurodegeneration. By semiquantitative scoring, the highest signals(fluorescence intensity and number of positive structures) were detectedin all mice dosed at 20 and 80 mg/kg (Table 6). Labelled structures werepresent at 8 mg/kg in 3 out of 4 mice, but to a clearly lower level thanat 20 and 80 mg/kg. Intravenous injection of 0.8 mg/kg did not result insignals above background with the applied method of visualization.Collectively, these data suggest that hC10-2_N32S antibodies in adose-dependent manner are able to penetrate into the brain parenchymaand specifically decorate targets in rTg4510 mice in vivo. FIG. 22(Panels A-D).

TABLE 6 Aged Tg4510 hC10-2_N32S Fluorscent Intensity (relative n = i.v.injection signal to 80 mg/kg) 3 80 mg/kg + in 3/3 +++ 4 20 mg/kg + in4/4 ++-+++ 4  8 mg/kg + in 3/4 + 4 0.8 mg/kg  + in 4/4 0Fluorescence-labelled tau structures detected in brain sectionsfollowing i.v. injection of hC10-2_N32S. Semiquantitative scoring: +++strong; ++ moderate; + weak; 0 not detected.

Example 11 Seed Tau Pathology in Tau Seed Preparation

Frozen cortical tissue samples from AD donors were obtained from TissueSolutions (Glasgow, UK). The brain tissue was weighed and homogenisedusing a knife homogeniser in sterile cold PBS (10 times volume of brainsample). The homogenate was then sonicated by 5×0.9 sec pulses on aBranson sonifier set at output 2. The homogenate was centrifuged at 3000g for 5 min at 4° C. and the supernatant was aliquoted, snap frozen ondry ice and stored at −80° C. until use.

Animals Used in Study

Transgenic mice expressing human mutated tau (P301L 0N4R) under atetracycline-controlled transactivator element in CamKII-positiveneurones (rtg4510) were used. This model normally begins to develop taupathology at 3-4 months of age. By feeding the mothers with doxycyclineduring pregnancy and for the first 3 weeks of the pup's life, thepathology develops at a later stage (after 6 months of age). Thedoxycycline pre-treated mice used in the studies were 2.5 months old atthe time of seeding.

Antibody Treatment

Antibodies were prepared in a concentration of 1.5 mg/ml in sterile PBSand made into aliquots with enough antibody for one dosing time-point.The aliquots were stored at 4° C. until used. From 2 months of age untilsacrifice at 5.5 months of age the mice received a weekly IP dose ofantibody (15 mg/kg equal to 10 ml/kg). Antibodies used: Control humanB12-IgGl, hC10-2, hC10-2_N32S and hC10-2_N32S_A101T.

Stereotactic Injection

Just after receiving the 3^(rd) antibody dose, mice were anesthetized byisoflourane inhalation and fixed in a stereotactic frame. The skull wasexposed and positioning adjusted until bregma and lambda were level. Ahole was drilled in the skull 2 mm lateral (right) and 2.4 mm posteriorto bregma. A 10 μl syringe with a 26 gauche, bevelled tip (SGE) was usedto inject the seeding material 1.4 mm ventral to the brain surface.

Two μl of material was slowly infused at the site (0.5 μl/min) and thesyringe was left thereafter for an additional 5 min before retraction.The wound was closed and sealed with sutures and mice were thermicallysupported during recovery from anesthesia. Mice were then housed for 3months before being perfusion-fixed with 4% paraformaldehyde.

Histology

Fixed brains were processed at Neuroscience Associates (Knoxville,Tenn.) using MultiBrain® technology. Up to 25 mouse brains were embeddedtogether per block and freeze-sectioned at 35 μm in the coronal planethrough the entire brain. Every 6^(th) section was stained with theGallyas silver stain to reveal neurofibrillary tangles. The sectionswere mounted, coverslipped and Gallyas silver-positive neurones (soma)were counted in the injected side of the hippocampi of all brains. Allsub-regions of the hippocampus were included. Eight sections werecounted per brain. Results reflect the sum of positive neurones from the8 sections.

Result

hC10-2, hC10-2_N32S and hC10-2_N32S_A101T all significantly reducedseeding of Tau tangles in the injected hippocampus (One way andova andTurkey's multiple comparisons test). hC10-2: 50%, hC10-2_N32S: 47% andhC10-2_N32S_A101T: 60% in comparison to control treated mice. FIG. 23.

1. A monoclonal antibody, or an epitope-binding fragment thereof,comprising: (a) a Light Chain CDR1 comprising an amino acid sequenceselected from the group consisting of SEQ ID NO:3, SEQ ID NO:31; SEQ IDNO:32; SEQ ID NO:33; SEQ ID NO:34; SEQ ID NO:35; SEQ ID NO:36; SEQ IDNO:37; SEQ ID NO:38; SEQ ID NO:40; and SEQ ID NO:46; (b) a Light ChainCDR2 comprising the amino acid sequence of SEQ ID NO:4; SEQ ID NO:41;and SEQ ID NO:47; (c) a Light Chain CDR3 comprising the amino acidsequence of SEQ ID NO:5; SEQ ID NO:42; and SEQ ID NO:48; (d) a HeavyChain CDR1 comprising the amino acid sequence of SEQ ID NO:6; SEQ IDNO:43; SEQ ID NO:49; SEQ ID NO:52; and SEQ ID NO:55; (e) a Heavy ChainCDR2 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:7; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQID NO:44; SEQ ID NO:50; SEQ ID NO:53; and SEQ ID NO:56; and (f) a HeavyChain CDR3 comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:8, SEQ ID NO:39; SEQ ID NO:45; SEQ ID NO:51; SEQID NO:54; and SEQ ID NO:57.
 2. A monoclonal antibody, or anepitope-binding fragment thereof, comprising: (a) a Light Chain selectedfrom the group consisting of SEQ ID NO:12; SEQ ID NO:16; SEQ ID NO:17;SEQ ID NO:18; SEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22 andSEQ ID NO:23; and (b) a Heavy Chain selected from the group consistingof SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:14; SEQ ID NO:15; SEQ ID NO:24;SEQ ID NO:25; SEQ ID NO:26; and SEQ ID NO:27.
 3. The monoclonalantibody, or epitope-binding fragment thereof, according to claim 1,wherein: (a) the Light Chain is SEQ ID NO:12; and (b) the Heavy Chain isselected from the group consisting of SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26 and SEQ ID NO:27.
 4. Themonoclonal antibody, or epitope-binding fragment thereof, according toclaim 1, wherein: (a) the Light Chain is selected from the groupconsisting of SEQ ID NO:16; SEQ ID NO:17; SEQ ID NO:18; SEQ ID NO:19;SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22 and SEQ ID NO:23; and (b) theHeavy Chain is SEQ ID NO:11.
 5. The monoclonal antibody, or epitopebinding fragment thereof, according to claim 1, comprising: (a) a LightChain CDR1 having the amino acid sequence of SEQ ID NO:31; (b) a LightChain CDR2 having the amino acid sequence of SEQ ID NO:4; (c) a LightChain CDR3 having the amino acid sequence of SEQ ID NO:5; (d) a HeavyChain CDR1 having the amino acid sequence of SEQ ID NO:6; (e) a HeavyChain CDR2 having the amino acid sequence of SEQ ID NO:7; and (f) aHeavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.
 6. Themonoclonal antibody, or epitope binding fragment thereof, according toclaim 1, comprising: (a) a Light Chain comprising the amino acidsequence of SEQ ID NO:16; and (b) a Heavy Chain comprising the aminoacid sequence of SEQ ID NO:11.
 7. The monoclonal antibody, or epitopebinding fragment thereof, according to claim 1, comprising: (a) a LightChain CDR1 having the amino acid sequence of SEQ ID NO:32; (b) a LightChain CDR2 having the amino acid sequence of SEQ ID NO:4; (c) a LightChain CDR3 having the amino acid sequence of SEQ ID NO:5; (d) a HeavyChain CDR1 having the amino acid sequence of SEQ ID NO:6; (e) a HeavyChain CDR2 having the amino acid sequence of SEQ ID NO:7; and (f) aHeavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.
 8. Themonoclonal antibody, or epitope binding fragment thereof, according toclaim 7, comprising: (a) a Light Chain comprising the amino acidsequence of SEQ ID NO:17; and (b) a Heavy Chain comprising the aminoacid sequence of SEQ ID NO:11.
 9. The monoclonal antibody, or epitopebinding fragment thereof, according to claim 1, comprising: (a) a LightChain CDR1 having the amino acid sequence of SEQ ID NO:33; (b) a LightChain CDR2 having the amino acid sequence of SEQ ID NO:4; (c) a LightChain CDR3 having the amino acid sequence of SEQ ID NO:5; (d) a HeavyChain CDR1 having the amino acid sequence of SEQ ID NO:6; (e) a HeavyChain CDR2 having the amino acid sequence of SEQ ID NO:7; and (f) aHeavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.
 10. Themonoclonal antibody, or epitope binding fragment thereof, according toclaim 9, comprising: (a) a Light Chain comprising the amino acidsequence of SEQ ID NO:18; and (b) a Heavy Chain comprising the aminoacid sequence of SEQ ID NO:11.
 11. The monoclonal antibody, or epitopebinding fragment thereof, according to claim 1, comprising: (a) a LightChain CDR1 having the amino acid sequence of SEQ ID NO:34; (b) a LightChain CDR2 having the amino acid sequence of SEQ ID NO:4; (c) a LightChain CDR3 having the amino acid sequence of SEQ ID NO:5; (d) a HeavyChain CDR1 having the amino acid sequence of SEQ ID NO:6; (e) a HeavyChain CDR2 having the amino acid sequence of SEQ ID NO:7; and (f) aHeavy Chain CDR3 having the amino acid sequence of SEQ ID NO:8.
 12. Themonoclonal antibody, or epitope binding fragment thereof, according toclaim 11, comprising: (a) a Light Chain comprising the amino acidsequence of SEQ ID NO:19; and (b) a Heavy Chain comprising the aminoacid sequence of SEQ ID NO:11.
 13. The monoclonal antibody, or epitopebinding fragment thereof, according to claim 1, comprising: (a) a LightChain CDR1 having the amino acid sequence of SEQ ID NO:3; (b) a LightChain CDR2 having the amino acid sequence of SEQ ID NO:4; (c) a LightChain CDR3 having the amino acid sequence of SEQ ID NO:5; (d) a HeavyChain CDR1 having the amino acid sequence of SEQ ID NO:6; (e) a HeavyChain CDR2 having the amino acid sequence of SEQ ID NO:7; and (f) aHeavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39.
 14. Themonoclonal antibody, or epitope binding fragment thereof, according toclaim 13, comprising: (a) a Light Chain comprising the amino acidsequence of SEQ ID NO:12; and (b) a Heavy Chain comprising the aminoacid sequence of SEQ ID NO:24.
 15. The monoclonal antibody, or epitopebinding fragment thereof, according to claim 1, comprising: (a) a LightChain CDR1 having the amino acid sequence of SEQ ID NO:31; (b) a LightChain CDR2 having the amino acid sequence of SEQ ID NO:4; (c) a LightChain CDR3 having the amino acid sequence of SEQ ID NO:5; (d) a HeavyChain CDR1 having the amino acid sequence of SEQ ID NO:6; (e) a HeavyChain CDR2 having the amino acid sequence of SEQ ID NO:7; and (f) aHeavy Chain CDR3 having the amino acid sequence of SEQ ID NO:39. 16-20.(canceled)
 21. The monoclonal antibody, or epitope binding fragmentthereof, according to claim 15, comprising: (a) a Light Chain comprisingthe amino acid sequence of SEQ ID NO:16; (b) a Heavy Chain comprisingthe amino acid sequence of SEQ ID NO:24.
 22. A pharmaceuticalcomposition comprising the monoclonal antibody, or epitope-bindingfragment thereof, according to claim 1 and a pharmaceutical acceptablecarrier
 23. A method for the treatment of a tauopathy, which comprisesadministering a therapeutically effective amount of the monoclonalantibody, or epitope-binding fragment thereof, according to claim 1 to asubject in need thereof, wherein said tauopathy is selected from thegroup consisting of Alzheimer's disease, Argyrophilic Grain Disease(AGD), Psychosis, particularly Psychosis due to AD or Psychosis inpatients with AD, apathy due to AD or apathy in patients with AD,psychiatric symptoms of patients with Lewy body dementia, ProgressiveSupranuclear Palsy (PSP), Frontotemporal dementia (FTD or variantsthereof), TBI (traumatic brain injury, acute or chronic), CorticobasalDegeneration (CBD), Picks Disease, Primary age-related tauopathy (PART),Neurofibrillary tangle-predominant senile dementia, Dementiapugilistica, Chronic traumatic encephalopathy, stroke, stroke recovery,neurodegeneration in relation to Parkinson's disease, Parkinsonismlinked to chromosome, Lytico-Bodig disease (Parkinson-dementia complexof Guam), Ganglioglioma and gangliocytoma, Meningioangiomatosis,Postencephalitic parkinsonism, Subacute sclerosing panencephalitis,Huntington's disease, lead encephalopathy, tuberous sclerosis,Hallervorden-Spatz disease and lipofuscinosis.
 24. A method for thetreatment of Alzheimer's disease which comprises administering atherapeutically effective amount of the monoclonal antibody, orepitope-binding fragment thereof, according to claim 1 to a subject inneed thereof.
 25. A method of diagnosing or imaging Alzheimer's diseaseor other tauopathies in a subject, said method comprising administeringto said subject a detectably labelled monoclonal antibody, orepitope-binding fragment thereof, according to claim
 1. 26. The methodaccording to claim 24, wherein the treatment is chronic.